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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- the dissection of the plastid genes and their expression mous amounts of particles make the method relatively effi- [21•–26•,27,28]. Some of these investigations focussed on cient [11••,19••,30]. Following protoplast isolation, a the function of several chloroplastic open reading frames simple protocol leads to a polyethylene glycol induced (ORFs) the function of which has remained obscure thus poration of cell membranes for the entry of genetic mater- far. After biolistic transformation of the giant ORFs ycf1 ial [34,36]. For unknown reasons, the technique has a and ycf2, all lines remained heteroplasmic despite repeated lower success rate than biolistic bombardment. cycles of regeneration under high selection pressure [21•]. Apparently, these genes encode products essential for cell Reporter gene strategies survival and are indispensable for chloroplast function. The insertion of reporter genes in the DNA construct that The same was concluded for ycf9 [22•], as selection on is integrated into the plastome, such as that coding for the spectinomycin did not result in homoplasmicity in spite of green fluorescent protein (GFP) [19••,20••,37] or resistance several selection cycles. In another study, however, homo- genes against lethal agents (e.g. spectinomycin and strepto- plasmicity was obtained and ycf9 knocked out [23•]. The mycin), is mandatory for tracing the transformed cells. ycf9 ORF was thought responsible for the production of small architectural components for the stabilization of Several new reporter gene strategies for the identification of light-harvesting complexes, in a similar way that the ycf6 plastome transformation have been developed over the past protein might contribute to the construction of the few years [20••,38•] (see Hohn, Levy and Puchta, this issue • cytochrome b6f complex [24 ]. A second focus point was pp 139-143). Vectors carrying the bacterial gene aphA-6, cod- the functioning of plastidic RNA. Emphasis was put on ing for an aminoglycoside phosphotransferase that detoxi- identification of the sites of action of endonucleases fies kanamycin or amikacin, were successfully inserted into 146 Plant biotechnology

Figure 1 selection medium, however, chimeric plants can develop in which visual distinction of the transgenic tissues is (a) impossible. The aforementioned fusion strategy lends visual support to the identification of the transplastomic leaf sectors and helps isolate sections of leaf that can be used for a second cycle of plant regeneration. (b) The inclusion of resistance marker genes has the disadvan- (c) tage that transformed cells must be traced by stringent methods. Plant regeneration from a cell embedded in the cellular environment killed by lethal agents is a risky under- taking and not fully understood. In the case of an optical marker like GFP, difficulties arise with the regeneration of a plant from a single GFP-expressing cell. Given the addi- tional problems of genetic contamination, the reporter gene strategy may not be the most ideal solution for selecting transformants. Therefore, alternative selection scenarios that eliminate the use of marker genes should be explored (see Hohn, Levy and Puchta, this issue pp 139-143).

1234 Injection of DNA material into chloroplasts A novel development is the injection of DNA material into (d) (e) (f) chloroplasts using syringes with extremely narrow tips [39••]. In animal cells, the nuclear injection of DNA through capillaries with tip diameters of one micron is common practice. The microinjection of DNA in plant cells has been less successful. The rigidity of the cell walls demands rigorous electrode penetration which inflicts con- siderable damage because of the turgor of the plant cell. Electrodes with smaller tips would cause less damage, but GEF-mediated genetic transformation. (a) Green fluorescent protein require extremely high pressures for fluid injection. A (GFP) expression in an epidermal cell of Vicia faba two days after novel microinjection principle combines a reduced tip size injection of a few plasmid constructs into the nucleus. (b) Cultures of Phormidium laminosum in BG-11 liquid medium containing ampicillin with the required injection force. Micropipettes with a tip grown for 30 days [39••]. Uninjected wild type (left); cells injected with diameter of 0.1 micron are drawn from capillaries of pARUB19 containing the bla gene conferring ampicillin resistance borosilicate or quartz glass, backfilled with a mixture of sil- (right). (c) Electrophoresis of PCR products of the ampicillin resis- icone oil and galinstan (an alloy of gallium, indium and tin) tance gene in P. laminosum: lane 1, uninjected wild type grown in the absence of ampicillin; lane 2, injected ampicillin-resistant culture; lane and sealed with a miniature glass cap. The fluid in the tip 3, PCR on pARUB19; lane 4, lambda DNA cut with BglI [39••]. is expelled by the pressure exerted by warming the mix- (d–f) Confocal laser scanning microscope images of GFP fluores- ture in the capillary. This galinstan expansion femtosy- cence (green) and chlorophyll autofluorescence (red) [39••]. ringe (GEF) allows injection of volumes in the femtolitre (d) Chlorophyll autofluorescence in a marginal mesophyll cell of a tobacco leaf. (e) GFP expression is clearly visible 24 h after injection range into nuclei. Injection of GFP constructs under the of a pNtcZ7 plasmid construct into the chloroplast. (f) Overlay of both control of the 35S promoter into nuclei of epidermal Vicia channels. (Parts of the figure were reproduced from [39••] with cells resulted in abundant expression of GFP (Figure 1a). permission from Nature Biotechnology.) The GEF tip is also suitable for insertion into chloroplasts [39••]. The injection of constructs containing the bla gene the plastome of Chlamydomonas [38•]. Transformants were into the filamentous cyanobacterium Phormidium laminosum identified by their survival on a selection medium. resulted in a stable transformation conferring ampicillin resistance (Figures 1b,c). Likewise, the injection of plasmid In a second novel strategy, the FLARE-S system, the DNA containing the gfp gene under the control of a chloro- aminoglycoside 3′′ adenyltransferase (aadA gene), which plast ribosomal RNA promoter [37] into chloroplasts of mar- confers resistance against spectomycin and streptomycin, ginal mesophyll cells of intact tobacco plants led to clearly is translationally fused to the gfp gene of Aequorea victoria visible expression of GFP in the chloroplasts (Figures 1d–f). [20••]. The drugs used in the selection procedure suppress The advantages of the procedure are obvious: the cells sur- chlorophyll production and inhibit shoot formation on vive the injection, the transformed cell can be spotted easi- plant regeneration media. The transformed lines can thus ly, and the cellular context remains intact. The latter fact be distinguished by their ability to form green shoots on permits the fate of the inserted gene or gene products to be bleached wild-type leaf sections. During cultivation on the followed. It appears that GFP moves to other chloroplasts in Novel approach in plastid transformation van Bel et al. 147

the injected cell. Because cell division in the leaf tissues in tool for improving the yield of transplastomic products. Also, question is thought to be fully completed at the time of the transfer of prefabricates through the plastid envelope injection, the division of chloroplasts is therefore unlikely that are further processed in the cytosol may be a target for and there is no evidence that cytosolic GFP is taken up by further development. chloroplasts [40,41]. Thus, GFP traffic between chloroplas- ts must have a different explanation. Acknowledgements JMH is supported by a Sir David Phillips Fellowship from the Macromolecular traffic between chloroplasts? Biotechnology and Biological Sciences Research Council. For the time being, this transfer is interpreted to occur through stromules, transient extensions of the chloroplast References and recommended reading envelope [42] which at first sight appear to be similar to bac- Papers of particular interest, published within the annual period of review, terial conjugation tubes. Similarly, tubular projections filled have been highlighted as: with GFP were observed in transplastomic tobacco plants • of special interest •• of outstanding interest [43•,44••]. Occasionally, tubules connecting chloroplasts are 1. Keeler KH, Turner CE, Bolick MR: Movement of crop transgenes seen in fusions between transplastomic and wild-type pro- into wild plants. In Herbicide Resistant Crops: Agricultural, toplasts [43•,45•]. The tubules are rare between chloroplas- Economic, Environmental, Regulatory and Technological Aspects. ts, but abundant between chloroplast-free plastids [45•]. So Edited by Duke SO. Boca Raton: CRC Press; 1995:303-330. far, the transfer of fluorescence was interpreted to be GFP 2. Daniell H, Datta R, Varma S, Fray S, Lee SB: Containment of herbi- cide resistance through genetic engineering of the chloroplast transport, but concomitant, interplastidic exchange of genome. Nat Biotechnol 1998, 16:345-348. genetic material may not at present be entirely ruled out. 3. Losey JE, Rayor LS, Carter ME: Transgenic pollen harms monarch •• larvae. Nature 1999, 399:214. Gene traffic between chloroplasts would presumably Apart from the containment risks imposed by pollen dissemination from Bt transgenomic plants, undesired side-effects of pollen-mediated Bt gene car- increase the chances and rate of a stable transformation riage emerge. This paper highlights deleterious effects of Bt transformation developing into a homoplasmic configuration and if the on nontarget insects feeding on milkweed leaves covered with Bt transgenic integrity of the cell material affects the extent of stromule corn pollen. formation, the development of transformation via GEF- 4. Stewart CN, Prakash CS: Chloroplast-transgenic plants are not a gene flow panacea. Nat Biotechnol 1998, 16:401. mediated injection may be advantageous. It is unclear 5. Cummins JE: Chloroplast-transgenic plants are not a gene flow whether the distribution of GFP in multiple plastids is relat- panacea. Nat Biotechnol 1998, 16:401. ed to the development of homoplasmicity and the stable 6. Bilang R, Potrykus I: Containing excitement over transplastomic transformation of chloroplasts following biolistic bombard- plants. Nat Biotechnol 1998, 16:333-334. ment [9,19••,20••]. How the development of homoplasmic- 7. Scott SE, Wilkinson MJ: Low probability of chloroplast movement ity would be affected by the movement of foreign genes •• from oilseed rape (Brassica napus) into wild Brassica rapa. Nat between the chloroplasts remains a matter of debate. Biotechnol 1999, 17:390-392. In view of the virtual absence of pollen-mediated chloroplast transfer, the risks Provided the reliability of the GEF method can be opti- of transgene dissemination via transplastomic pollen of oilseed rape mized, the necessity of reporter gene inclusion may disap- (Brassica napus) are considered to be negligible. The chance of transgene introgression is demonstrated to be low. On the basis of these investigations, pear as the location of the cell injected is known. In the containment of transplastomic genes seems to be likely for oilseed rape. addition, the GEF promises to provide an invaluable tool to 8. Chèvre AM, Eber F, Baranger A, Renard M: Gene flow from trans- investigate plastid gene expression in vivo and also informa- genic crops. Nature 1997, 389:924. tion exchange between plastids occupying the same cell. 9. Staub JM, Maliga P: Long regions of homologous DNA are incor- porated into the tobacco plastid genome by transformation. Plant Conclusions Cell 1992, 4:39-45. Although considerable progress has been made over the 10. Staub JM, Garcia B, Graves J, Hajdukiewicz PTJ, Hunter P, Nehra N, •• Paradkar V, Schlittler M, Carroll JA, Spatola L et al.: High-yield pro- past few years, there is a number of hurdles to overcome duction of a human therapeutic protein in tobacco chloroplasts. before plastome transformation becomes a very attractive Nat Biotechnol 2000, 18:333-338. Transgenic plants may become low-cost sources for large amounts of precious device for plant biotechnology. Firstly, the number of plant proteins. Transplastomic technology may amplify this potential because of an species to which plastome technology is applicable needs increase in biological containment and high expression rates. Transplastomic to be increased considerably; thus far, only a limited num- tobacco plants were able to produce massive amounts of a therapeutic pro- tein, human somatropin, which is used in the treatment of hypopituitary ber of culture plants has been transformed successfully by dwarfism and is envisaged to be useful in other medical applications. plastome manipulation. Secondly, the success rate of gene 11. Guda C, Lee SB, Daniell H: Stable expression of a biodegradable insertion into the plastome has to be increased; this may •• protein-based polymer in tobacco chloroplasts. Plant Cell Rep 2000, 19:257-262. reduce the need for extensive selection procedures. Transplastomic technology may provide a basis for the future mass produc- Thirdly, the screening protocols must be simplified and tion of a wide range of complex materials (see also [10••]). A high yield of become applicable to a large range of plant species [46]. bioelastic protein-based polymers (100-fold that in transgenic plants) was obtained with transplastomic tobacco plants carrying an artificial gene encoding a repetitive sequence observed in all sequenced mammalian Plastome technology may be potentiated by several novel elastin proteins. strategies. The stromule-mediated interplastidic migration 12. Svab Z, Harper EC, Jones JDG, Maliga P: Aminoglycoside-3′′- adenyltransferase confers resistance to spectinomycin and of macromolecules, which in itself is an intriguing event from streptomycin in Nicotiana tabacum. Plant Mol Biol 1990, a fundamental point of view, may be used as an additional 14:197-205. 148 Plant biotechnology

13. Daniell H: New tools for chloroplast genetic engineering. Nat conditions, the growth rate of the transformants is significantly lower. The gene Biotechnol 1999, 17:855-856. product of ycf9 is part of the light-harvesting antenna of photosystem II, which explains a reduced photon capture efficiency under low-light conditions. 14. McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P: Amplification of a chimeric Bacillus gene in chloroplasts leads to 24. Hager M, Biehler K, Illerhaus J, Ruf S, Bock R: Targeted inactivation an extraordinary level of an insecticidal protein in tobacco. • of the smallest plastid genome-encoded open reading frame Biotechnology 1995, 13:362-365. reveals a novel and essential subunit of the cytochrome b6f com- plex. EMBO J 1999, 18:5834-5842. 15. Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ: •• The smallest conserved ORF in the tobacco plastome, ycf6, encodes a Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in polypeptide of only 29 amino acids. The ycf6 (now called petN) gene prod- chloroplasts confers resistance to plants against susceptible and uct turned out to be an essential building block or stabilization element of the Bt-resistant insects. Proc Natl Acad Sci USA 1999, 96:1840-1845. cytochrome b6f complex and is therefore indispensible for the transfer of These transplastomic plants are killer variants to earlier transplastomic Bt electrons between photosystems II and I. plants (see also [14]): the mortality factor is several orders of magnitude higher. These plants are expected to overcome the ready development of Bt 25. Hermann M, Bock R: Transfer of plastid RNA-editing activity to resistance in the field. • novel sites suggests a critical role for spacing in editing-site recognition. Proc Natl Acad Sci USA 1999, 96:4856-4861. 16. Lee HJ, Lee SB, Chung JS, Han SU, Han O, Guh JO, Jeon JS, An G, •• The mechanism of RNA editing by producing mRNA through C-to-U con- Back K: Transgenic rice plants expressing a Bacillus subtilis pro- versions is unknown. A crucial question is how the cytidine residues to be toporphyrinogen oxidase gene are resistant to diphenyl ether altered are recognised by the guide RNA. This paper indicates that the iden- herbicide oxyfluorfen. 41 Plant Cell Physiol 2000, :743-749. tity of some editing sites in the plastome is defined by their distance from a Transplastomic rice plants expressing the Bacillus subtilis Protox gene show certain upstream sequence element. better resistance to oxyfluorfen than transgenomic plants. This is a new approach to the selective killing of weeds in paddies by use of oxyfluorfen, 26. Rott R, Liveanu V, Drager RG, Higgs D, Stern DB, Schuster G: an inhibitor of chlorophyll synthesis. • Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast RNA affects 3′ end maturation 17. Kavanagh TA, Thanh ND, Lao NT, McGrath N, Peter SO, Horvath EM, in vitro but not in vivo. Plant Mol Biol 1999, 40:679-686. •• Dix PJ, Medgyesy P: Homologous plastid DNA transformation in The processing of plastidic pre-mRNA involves endonucleolytic cleavage tobacco is mediated by multiple recombination effects. Genetics encoded by codons several nucleotides downstream of stem–loop struc- 1999, 152:1111-1122. tures. In Chlamydomonas chloroplasts, the mature 3′ end is generated by Are transformation vectors for tobacco also suitable for other species or is it cleavage at an AU-rich site located about ten nucleotides from the stem necessary to construct specific transformation vectors for each species? In loop. Mutations of this site obtained by biolistic transformation indicate that view of future developments, this is a major issue in transplastomics for the the misinformation can be overcome in vivo. sake of efficiency. Insertion of homeologous (incompletely homologous) Solanum plastid DNA into the Nicotiana plastome showed a transformation 27. Eibl C, Zou ZR, Beck A, Kim M, Mullet J, Koop HU: In vivo analysis frequency comparable with that obtained with homologous DNA. It therefore of plastid psbA, rbcL and rpl32UTR elements by chloroplast seems likely that within certain limits of nucleotide sequence, which have as transformation: tobacco plastid gene expression is controlled by yet to be determined, plastid transformation vectors do not need to be entirely modulation of transcript levels and translation efficiency. Plant J species-specific. 1999, 19:333-345. 18. Sidkar SR, Serino G, Chaudhuri S, Maliga P: Plastid transformation 28. De Santis-Maciossek G, Kofer W, Bock A, Schoch S, Maier RM, in Arabidopsis thaliana. Plant Cell Rep 1998, 18:20-24. Wanner G, Rüdiger W, Koop HU, Herrmann RG: Targeted disrup- 19. Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PTJ, Staub JM, tion of the plastid RNA polymerase genes rpoA, B and C1: molec- •• Nehra NS: Stable chloroplast transformation in potato: use of ular biology, biochemistry and ultrastructure. Plant J 1999, green fluorescent protein as a plastid marker. Plant J 1999, 18:477-489. 19:209-216. 29. Bogorad L: Engineering chloroplasts: an alternative site for for- Markers and regeneration methods leading to high efficiency in the plastid eign genes, proteins, reactions and products. Trends Biotechnol transformation of tobacco were applied to potato and resulted in a stable 2000, 18:257-263. transformation. Cotransfer of aadA (spectinomycin/streptomycin resistance) and GFP in a pZS192 plasmid construct allowed the visual detection of the 30. Tomizawa K, Shikanai T, Shomoide A, Foyer CH, Yokota A: Revertant transformed chloroplasts. of no-active Rubisco tobacco mutant, Sp25, obtained by chloro- plast transformation method using microprojectile bombardment. 20. Khan MS, Maliga P: Fluorescent antibiotic resistance marker for •• Advances in Chemical Conversions for Mitigating Carbon Dioxide tracking plastid transformation in higher plants. Nat Biotechnol 1998, 114:609-612. 1999, 17:910-915. Using pMSK56 or pMSK57 plasmids as vectors for a GFP–aadA fusion 31. Kanevski I, Maliga P, Rhoades DF, Gutteridge S: Plastome engineering construct, transplastomic plants produced a bifunctional marker gene prod- • of ribulose-1,5-bisphosphate carboxylase/oxygenase in tobacco uct (FLARE) that combines antibiotic resistance with optical tracking. Such to form a sunflower large subunit and tobacco small unit hybrid. a gene product is very useful when resistant plant areas are not readily rec- Plant Physiol 1999, 119:133-141. ognizable. This novel method is an important step toward plastid transfor- After chloroplast transformation with the rbcL gene from either sunflower mation of monocots where transformation is not associated with a (Helianthus annuus) or the cyanobacterium Synechococcus, three stable distinguishable tissue culture phenotype. lines of tobacco were obtained. As outlined in detail in the text, mutagene- sis resulted in different forms of the rbcL gene encoding chimeric enzymes The two largest 21. Drescher A, Ruf S, Calsa T, Carrer H, Bock R: that are active in vivo. • chloroplast genome-encoded open reading frames of higher plants are essential genes. Plant J 2000, 22:97-104. 32. Whitney SM, von Caemmerer S, Hudson GS, Andrews TJ: Directed Mutant alleles for targeted disruption and/deletion of two plastidic giant • mutation of the Rubisco large subunit of tobacco influences pho- ORFS (ycf1 and ycf2) were introduced into the tobacco plastid genome. As torespiration and growth. Plant Physiol 1999, 121:579-588. all transformed lines remained heteroplastomic after the harshest selection After biolistic bombardment with pL335V plasmids carrying a modified (by treatments, the gene products appear to be essential for cell survival. changing codon 335 to encode valine instead of leucine) gene for rbcL, the tobacco plant transformants showed reduced Michaelis constants for CO , 22 Mäenpää P, Gonzalez EB, Chen L, Khan MS, Gray JS, Aro EM: The 2 • ycf-9 (ORF-62) gene in plant chloroplast genome encodes a O2 and ribulose-1,5-bisphosphate and correspondingly lower relative growth rates. The new strategy overcomes earlier problems in the mutagen- hydrophobic protein of stromal thylakoid membranes. J Exp Bot esis of higher plant Rubisco. 2000, 51:375-382. Among the plastid gene products essential for chloroplast functioning is a 33. Daniell H, Vivekananda J, Nielsen BL, Ye GN, Tewari KK, Sanford JC: hydrophobic protein of stromal thylakoid membranes encoded by the ycf9 Transient foreign gene expression in chloroplasts of cultured (ORF 62) gene. This was inferred by the finding that transplastomic tobac- tobacco cells after biolistic delivery of chloroplast vectors. Proc co plants carrying plasmid constructs inactivating the gene with a selectable Natl Acad Sci USA 1990, 87:88-92. marker cassette never reached homoplasticity. 34. O’Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P: Chloroplast 23. Ruf S, Biehler K, Bock R: A small chloroplast-encoded protein as a transformation in plants: polyethylene glycol (PEG) treatment of • novel architectural component of the light harvesting antenna. protoplasts is an alternative to biolistic delivery systems. Plant J J Cell Biol 2000, 149:369-377. 1993, 3:729-738. Homoplasmic ycf9 tobacco plants displayed no mutant phenotype under stan- dard greenhouse conditions. This seems to contradict the conclusion (see 35. Koop HU, Steinmüller K, Wagner H, Röβler C, Eibl C, Sacher L: [22•]) that ycf9 is essential for chloroplast functioning. Under low-level light Integration of foreign sequences into the tobacco plastome via Novel approach in plastid transformation van Bel et al. 149

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