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Kanamycin resistance as a selectable marker for plastid transformation in tobacco
Abstract
We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 micrograms/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 micrograms/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10,000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.
... Resulta casi anecdótico la utilización de otros genes marcadores de selección como los de detoxificación de kanamicina (Carrer et al., 1993;Huang et al., 2002;Kumar et al., 2004a), detoxificación de la betaína aldehído por la acción de la betaína aldehído deshidrogenasa (BADH) y la selección de transformantes plastidiales empleando el herbicida fosfinotricina y el gen bar (Staub et al., 2002;Sidorov et al., 2008) o el glifosato y el gen CP4 (Staub et al., 2002). ...
... El sistema nptII-kanamicina es uno de los más empleados en la transformación de plantas tanto en dicotiledóneas como tabaco y Arabidopsis como en monocotiledóneas como arroz y maíz (Miki y McHugh, 2004). No se conocían mutaciones espontáneas que pudieran dar lugar a la aparición de escapes (Carrer et al., 1993;Huang et al., 2002) hasta que se intentó transformar plastidialmente la alfalfa. En esta especie, no se obtuvieron plantas transplastómicas, pero sí escapes resistentes que presentaron una mutación en el gen 16S ARNr y cuyo fenotipo era anormal y presentaban un crecimiento más lento (Rosellini et al., 2004). ...
... El empleo de este sistema de selección en transformación plastidial es mucho menos frecuente que el sistema espectinomicina/estreptomicina y el gen aadA. Se ha demostrado que la enzima NPTII es más eficiente detoxificando kanamicina cuando es expresada en el citosol que en el plastidio (Carrer et al., 1993). Sin embargo ha servido para transformar hojas y protoplastos de tabaco (Carrer et al., 1993;Carrer y Maliga, 1995;Huang et al., 2002;Stegemann y Bock, 2009), Chlamydomonas (Bateman y Purton, 2000) y algodón (Kumar et al., 2004a). ...
Maize is one of the most important crops for the nutritional intake of humans, being such via direct utilization as food or through livestock breeding. The constant growth of human population, the increasing meat consumption in emerging economy countries, the use of grain for biofuel production or the climate change are demanding an increase in the productivity of this crop. Plant biotechnology offers diverse procedures to achieve this objective, being plastid transformation one of them. This technique brings important advantages to plant transformation such as high expression level of the gene of interest or prevent the transgene transmission through pollen to other crops due to the maternal inheritance of plastids. Achieving plastid transformation of maize would allow the transfer of those advantages to this important crop.
We have developed diverse in vitro culture procedures and worked with different maize genotypes to select the best candidates to be plastidially transformed. The best protocol tested has been the regeneration through somatic embryogenesis induced from immature embryos. We also obtained a green calli culture procedure using the peptide hormone -PSK, designed a second round of regeneration on selective media using mature somatic embryos and a protocol for culturing callus cell aggregates on liquid media as an alternative to cell suspensions.
The three selection schemes tested were antibiotic based: the gene aadA gene and streptomycin, the nptII gene and kanamycin and the Aph(4) gene and hygromycin selection. Those genes were cloned in four different plastid transformation vectors which introduced the transgenes into the following regions of the inverted repeat regions of the plastome: 16SRNAr-trnV-ORF85/ORF58 or 16SRNAr-trnI/trnA-23SRNAr. A total of 54 transformation experiments were made with 12 different types of materials and 1738 maize regenerants were obtained, 7 of which were transplastomic regenerants with a high level of heteroplasmy. Six of them were obtained in streptomycin selection and 1 with hygromycin.
It was also designed an alternative selection scheme which was based on the complementation of 3 nuclear photosynthetic albino mutants: csr1-1, crs1-2 y crs2-2. We obtained embryogenic calli from those mutants cultured in light or dark conditions from immature embryos. The mutated gene was introduced in a plastid transformation vector which introduces the gene into the 16SRNAr-trnI/trnA-23SRNAr recombination zone. No regenerants were obtained from 6 transformation experiments.
... The AIBW expression cassette contains the Prrn promoter fused to the tobacco rbcL Shine-Dalgarno sequence, aadA gene, G10L leader, gfp gene and rrnBT, followed by the clpP promoter and 5′UTR from Zea mays (PclpP) 40 , BCTV Rep gene and psbA 3′UTR (TpsbA) from tobacco (Table 1 and Fig. 3a; GenBank accession number MZ074310). The AIDR cassette consists of the Prrn promoter fused to the tobacco rbcL Shine-Dalgarno sequence, gfp reporter gene, G10L leader, nptII (neomycin phosphotransferase II) selectable marker 41 and tobacco TpsbA (Table 1 and Fig. 5a; GenBank accession number MZ074311). The AJWY transgene was cloned in a pGreenII-0029 (ref. ...
... Leaves from three-to four-leaf stage tobacco plants grown in soil under standard greenhouse conditions (Supplementary Methods) were sterilized and bombarded with 0.6-μm gold particles coated with vector DNA using a helium particle gun (PDS-1000/ He; Bio-Rad). Primary transformants were selected on Murashige and Skoog sucrose medium supplemented with 500 mg l -1 spectinomycin for aadA-mediated selection 36 or with 100 mg l -1 kanamycin for nptII-mediated selection 41 . For nuclear transformation with the AJWU construct, the Agrobacterium-mediated method was used 43 and primary transformants were selected on Murashige and Skoog sucrose medium supplemented with 300 mg l -1 kanamycin. ...
Plant molecular farming, that is, using plants as hosts for production of therapeutic proteins and high-value compounds, has gained substantial interest in recent years. Chloroplasts in particular are an attractive subcellular compartment for expression of foreign genes. Here, we present a new method for transgene introduction and expression in chloroplasts that, unlike classically used approaches, does not require transgene insertion into the chloroplast genome. Instead, the transgene is amplified as a physically independent entity termed a ‘minichromosome’. Amplification occurs in the presence of a helper protein that initiates the replication process via recognition of specific sequences flanking the transgene, resulting in accumulation of extremely high levels of transgene DNA. Importantly, we demonstrate that such amplified transgenes serve as a template for foreign protein expression, are maintained stably during plant development and are maternally transmitted to the progeny. These findings indicate that the minichromosome-based approach is an attractive tool for transgene expression in chloroplasts and for organelle genome engineering. A new method for transgene expression in chloroplasts is developed, which amplified transgenes efficiently as a minichromosome. Such amplified transgenes can express foreign proteins and are maintained stably during plant development and inheritance.
... Tobacco was chosen for small seed size (ca. 700 lm in length) and the availability of the transgenic line, Havana PT (pTNH32-70-2, a gift from P. Maliga), carrying a functional kanamycin antibiotic resistance gene (nptII) in the chloroplasts (Carrer et al., 1993). Chloroplasts, which are derived from ancient bacteria, are photosynthetic, intracellular organelles with simple genomes; they are numerous in plant cells, providing high copy numbers of the model gene. ...
... The lack of mutant phenotypes in the survivors (and in their progeny) of full space exposure in EXPOSE-E (Fig. 1A) raised the question of whether DNA damage had occurred. Quantification of chloroplast DNA damage in tobacco seeds from EXPOSE-E, using a bacterial kanamy-cin resistance gene (nptII) inserted into the chloroplast genome (Carrer et al., 1993), revealed structural and functional damage correlated with exposure to UV light. ...
For life to survive outside the biosphere, it must be protected from UV light and other radiation by exterior shielding or through sufficient inherent resistance to survive without protection. We tested the plausibility of inherent resistance in plant seeds, reporting in a previous paper that Arabidopsis thaliana and tobacco (Nicotiana tabacum) seeds exposed for 558 days outside the International Space Station (ISS) germinated and developed into fertile plants after return to Earth. We have now measured structural genetic damage in tobacco seeds from this EXPOSE-E experiment by quantitatively amplifying a segment of an antibiotic resistance gene, nptII, inserted into the chloroplast genome. We also assessed the survival of the antibiotic resistance encoded by nptII, using marker rescue in a soil bacterium. Chloroplast DNA damage occurred, but morphological mutants were not detected among the survivors. In a second, longer mission (EXPOSE-R), a nearly lethal exposure was received by Arabidopsis seeds. Comparison between a ground simulation, lacking UV<200nm, and fully exposed seeds in space indicated severe damage from these short wavelengths and again suggested that DNA degradation was not limiting seed survival. To test UV resistance in long-lived, larger seeds, we exposed Arabidopsis, tobacco, and morning glory seeds in the laboratory to doses of UV254nm, ranging as high as 2420 MJ m(-2). Morning glory seeds resisted this maximum dose, which killed tobacco and Arabidopsis. We thus confirm that a naked plant seed could survive UV exposures during direct transfer from Mars to Earth and suggest that seeds with a more protective seed coat (e.g., morning glory) should survive much longer space travel. Key Words: UV light-Flavonoids-Sinapate-DNA degradation-Arabidopsis-Tobacco-Seeds-Space-International Space Station-EXPOSE-E-EXPOSE-R. Astrobiology 17, xxx-xxx.
... Every vector must contain 2 x 500-1,000 bp homologous sequences, e.g., amplified from the host by PCR and interrupted by a resistance cassette (e.g., the kanamycin cassette KanR used here) 37,38 . For expression, a promoter, coding sequence (e.g., for sfGFP 39 ), terminator, and resistance cassette must be cloned between the homologous sequences. ...
... These antibiotics function by binding to the chloroplast 30S ribosomal subunit and inhibiting protein translation [58][59][60]. Other resistance markers that have been successfully used in plastid transformation are the nptII(neo) gene and cat gene conferring resistance against neomycin/kanamycin and chloramphenicol antibiotics, respectively [61,62]. Since the use of antibiotics as a selection marker in commercial transgenic crop plants can cause a problem with approval from the regulatory authorities, metabolism-specific genes such as betaine aldehyde dehydrogenase gene (BADH) can be used as selection markers. ...
Chloroplasts are specialized organelle that are responsible for converting light energy to chemical energy, thereby driving the carbon dioxide fixation. Apart from photosynthesis, chloroplast is the site for essential cellular processes that determine the plant adaptation to changing environment. Owing to the presence of their own expression system, it provides an optimum platform for engineering valued traits as well as site for synthesis of bio-compounds. Advancements in technology have further enhanced the scope of using chloroplast as a multifaceted tool for the biotechnologist to develop stress-tolerant plants and ameliorate environmental stress. Focusing on chloroplast biotechnology, this review discusses the advances in chloroplast engineering and its application in enhancing plant adaptation and resistance to environmental stress and the development of new bioproducts and processes. This is accomplished through analysis of its biogenesis and physiological processes, highlighting the chloroplast engineering and recent developments in chloroplast biotechnology. In the first part of the review, the evolution and principles of structural organization and physiology of chloroplast are discussed. In the second part, the chief methods and mechanisms involved in chloroplast transformation are analyzed. The last part represents an updated analysis of the application of chloroplast engineering in crop improvement and bioproduction of industrial and health compounds.
... Every vector must contain 2 x 500-1,000 bp homologous sequences, e.g., amplified from the host by PCR and interrupted by a resistance cassette (e.g., the kanamycin cassette KanR used here) 37,38 . For expression, a promoter, coding sequence (e.g., for sfGFP 39 ), terminator, and resistance cassette must be cloned between the homologous sequences. ...
Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.
... The aadA gene is the most widely used and successful marker gene in the plastid transformation field allowing antibiotic selection (spectinomycin and streptomycin) of transplastomic microalgae [4] and flowering plants [1]. Other antibiotic/marker gene combinations used to select plastid transformants include kanamycin/nptII [5], kanamycin/aphA6 [6], chloramphenicol/cat [7], and tobramycin/aac6-aphA2 [8]. Removal of marker genes conferring antibiotic resistance is particularly important for gaining the regulatory approval needed for commercialization of transplastomic crops. ...
Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.
... In the transformation of plant chloroplasts, other selection marker genes have also been reportedly used for the selection of transformed cells. In plants, the following genes have been reported: nptII gene (neomycin) [44], aphA-6/nptII genes (kanamycin) [45], hpt gene (hygromycin) [5], aac(6 0 )-Ie/aph(2 00 )-Ia gene (tobramycin and gentamicin) [46], aphA-6 gene (amikacin) [45], and cat gene (chloramphenicol) [47]; in algae, the following genes have been reported: aphA-6 gene (amikacin) [48], cat gene (chloramphenicol) [49], ble gene (zeocin) [50], and ereB gene (erythromycin) [51]. ...
Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology’s high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.
How to cite: Quintín Rascón-Cruz Q, González-Barriga CD, Iglesias-Figueroa BF, et al. Plastid transformation: Advances and challenges for its implementation in agricultural crops. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.005
... La lectura de algunas revisiones puede dar la idea de que en el campo de la Por lo que respecta a genes marcadores que confieren resistencia a antibióticos, los dos más utilizados siguen siendo el gen nptII (Fraley et al., 1983, Carrer et al., 1993 y el gen hpt o hph (Waldron et al., 1985). (Gonzalez et al., 1978). ...
Cucumber is one of the most important horticultural species at economic level. Despite its agronomic relevance, there is currently no efficient transformation method available. The problems to obtain transgenic cucumber plants are mainly due to the low morphogenetic response in explants of certain genotypes, poor adequacy of selection methods, decrease in regeneration rate as a consequence of the usual treatments in a transformation experiment and, above all, the high rate of 'escape' plants. Taking into account the existing problems, the first objective of the present work has been the evaluation of the morphogenetic response in cucumber explants and the development of regeneration methods potentially useful in transformation experiments. These studies have been carried out with two commercial cultivars, four pure lines and one line that has been frequently used in cucumber breeding programs. Different types of primary explants have been evaluated to determine which may be the most suitable in a transformation experiment. The influence of the ontogenic state of the seedling on the degree of myxoploidy in cotyledon explants has been studied. Likewise, the ploidy level of regenerated plants from these explants has been determined. Regarding the culture medium, the effect of growth regulators, as well as that of other components, such as silver nitrate or copper sulphate, has been studied. In the context of the second objective, studies on the different stages of the transformation process by co-cultivation of cucumber explants with Agrobacterium tumefaciens have been carried out. Several factors, such as the Agrobacterium strain, how to carry out the phases of inoculation and co-culture, the suitability of different marker genes in the selection process and the influence of different components of the culture medium have been analyzed. Attempts have also been made to infer the causes of the occurrence of 'escape' plants or the formation of chimeras in cucumber transformation experiments. Finally, based on the results obtained, some solutions that could help to avoid this problem in future experiments are proposed.
... Boynton, Gillham, and colleagues first achieved plastid transformation of Chlamydomonas with this plastid promoter in 1988 [40]; Svab, Hajukiewicz and Maliga followed, in 1990, with the first report of plastid transformation in tobacco, although they used a different fragment of chloroplast DNA in their target plasmid [50]. Plastid transformation has since been reported in many flowering plants, such as tobacco [50,51,52], Arabidopsis [53,54], potato [55], rice [56], rapeseed [57], and tomato [58]. A protocol for plastid transformation of an elite rapeseed cultivar (B. ...
In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.
... The application of aadA as a selection marker in combination with a selection agent, spectinomycin or streptomycin, for the development of the plastid transformation system was key (Khan 2012;Svab and Maliga 1993). Although other selection markers such as kanamycin and chloramphenicol resistant genes have been used, their application was not as successful as that of aadA in plastid transformation model tobacco (Carrer et al. 1993;Li et al. 2011). In addition, herbicideresistance genes such as bar, EPSPS, or mutated acetolactate synthase (Shimizu et al. 2008;Ye et al. 2001) were not sufficiently effective for primary selection but could be used in secondary selection procedures (Daniell et al. 1998;Lutz et al. 2001). ...
Plastid transformants form biofactories that are able to produce extra proteins in plastids when they are in a homoplasmic state. To date, plastid transformation has been reported in about twenty plant species; however, the production of homoplasmic plastid transformants is not always successful or easy. Heteroplasmic plants that contain wild-type plastids produce fewer target proteins and do not always successfully transfer transgenes to progeny. In order to promote the generation of homoplasmic plants, we developed a novel system using barnase–barster to eliminate wild-type plastids from heteroplasmic cells systematically. In this system, a chemically inducible cytotoxic barnase under a plastid transit signal was introduced into nuclear DNA and barster, which inhibits barnase, was integrated into plastid DNA with the primary selection markers aminoglycoside 3′-adenylyltransferase (aadA) and green fluorescence protein (GFP) gene. As expected, the expression of the plastid barnase was lethal to cells as seen in leaf segments, but barster expression in plastids rescued them. We then investigated the regeneration frequency of homoplasmic shoots from heteroplasmic leaf segments with or without barnase expression. The regeneration frequency of homoplasmic-like shoots expressing barnase–barster system was higher than that of shoots not expressing this. We expect that the application of this novel strategy for transformation of plastids will be supportive to generate homoplasmic plastid transformants in other plant species.
... For selection in plastid transformation, aadA gene conferring spectinomycin resistance is widely used and is the most efficient selective marker [2]; however, nptII gene conferring kanamycin resistance has also been developed for tobacco plastid transformation [34]. Thus, besides the selection marker aadA gene which has been used in pCEV1, pCEV2, pCEV4, and their derivatives, we also constructed nptII gene as a selection marker for tomato plastid transformation. ...
Plastid transformation has a number of advantages in comparison with nuclear transformation. Currently, only tobacco (Nicotiana tabacum) is routinely used in plastid transformation. Here we constructed a series of chloroplast expression vectors specific for spinach (pCEV1), tomato (pCEV2 and pCEV3), and N. benthamiana (pCEV4). Selection marker aminoglycoside 3'-adenyltransferase (aadA) conferring spectinomycin resistance was used in pCEV1, pCEV2, and pCEV4, while selection marker neomycin phosphotransferase II (nptII) was used in pCEV3. The expression cassette in these vectors was integrated in the intergenic spacer between trnI and trnA of plastid genome via homologous recombination. Several transgenes, including a reporter gene encoding GFP:GUS fusion protein and genes from tomato (lycopene beta-cyclase, zeta-carotene desaturase) and bamboo mosaic virus satellite RNA (encoding coat protein CP20), were independently cloned into some of these vectors. Transient GUS expression was detected in spinach leaves bombarded by pCEV1/GFP-GUS. Functional expression of selection markers aadA and nptII was demonstrated for spinach, tomato, and N. benthamiana. Seedling assay from T0 self-pollinated plant of transplastomic N. benthamiana confirmed maternal inheritance of transgenes, and genomic PCR analysis confirmed integration of transgenic expression cassette into the plastid genome of N. benthamiana. Moreover, auxiliary vectors pECaad and pECnpt are also reported.
... The choice of selectable marker genes is significant for successful and efficient plastid transformation and among which aminoglycoside 3′-adenylyltransferase (aadA) for spectinomycin resistance is commonly used. A few alternative selectable markers have been extensively employed apart from aadA that includes, chimeric nptII genes encoding neomycin phosphotransferase, conferring resistance to kanamycin (Carrer et al. 1993), aphA-6 encoding an aminoglycoside phosphotransferase that confers resistance to kanamycin (Huang et al. 2002), and cat gene-encoding chloramphenicol acetyltransferase for chloramphenicol resistance (Li et al. 2011). Very recently, another selectable marker aminoglycoside acetyltransferase (6′)-Ie/aminoglycoside phosphotransferase (2″) gene was employed which confers resistance to tobramycin as the replacement of aadA in transplastomic selection (Tabatabaei et al. 2017). ...
In the present study, we focused on designing a species-specific chloroplast vector for Capsicum annuum L. and finding out its transformation efficiency compared to a heterologous vector. The plastid transformation vector (CaIA) was designed to target homologous regions trnA and trnI of IR region. A selectable marker gene aadA, whose expression is controlled by psbA promoter and terminator, was cloned between two flanking regions. A heterologous vector pRB95, which targets trnfM and trnG of LSC region along with aadA driven by rrn promoter and psbA terminator, was also used for developing plastid transformation in Capsicum. Cotyledonary explants were bombarded with stabilized biolistic parameters: 900 psi pressure and 9 cm flight distance, and optimized regeneration protocol (0.7 mg/L TDZ + 0.2 mg/L IAA) was used to obtain transplastomic lines on selection medium (300 mg/L spectinomycin). The aadA integration and homoplasmy were confirmed by obtaining 1.2 and 3.7 kb amplicons in CaIA transformants and subsequently verified by Southern blotting, whereas in pRB95 transformants, integration was confirmed by PCR with 1.45 kb and 255 bp amplicons corresponding to aadA integration and flanks, respectively. The transformation efficiencies attained with two plastid vectors were found to be 20%, i.e., 10 transplastomic lines in 50 bombarded plates, with CaIA and 2%, i.e., 1 transplastomic line in 50 bombarded plates, with heterologous pRB95, respectively.
... A number of selectable markers are employed in attaining stable plastid transformants that includes the aadA gene (encodes aminoglycoside 3′-adenylyltransferase) that confers resistance to spectinomycin (Goldschmidt-Clermont 1991). Other selectable marker genes used in plastid transformation are nptII (Carrer et al. 1993), codA (Serino and Maliga 1997), bar (Iamtham and Day 2000), EPSPS (Ye et al. 2001), gfp (Khan and Maliga 1999), and uidA (Staub and Maliga 1995). ...
... This is likely due to the high enzymatic activity of the AadA protein and the high specificity of spectinomycin as a potent inhibitor of plastid translation. A few alternative selectable markers have been developed for tobacco plastid transformation, including the nptII gene encoding a neomycin phosphotransferase that confers resistance to kanamycin (Carrer et al. 1993), the aphA-6 gene that encodes an aminoglycoside phosphotransferase also conferring kanamycin resistance (Huang et al. 2002), and the cat gene encoding chloramphenicol acetyltransferase and conferring resistance to chloramphenicol (Li et al. 2011). However, due to their substantially lower efficiency than aadAbased selection, they have not become widely adopted. ...
... Finally, a second kanamycin resistance gene has been developed for plant chloroplasts. The neomycin phosphotransferase gene, nptII, works as a selectable marker in tobacco, albeit with lower efficiency than the aphA-6 marker [37,44]. In cotton, a higher transformation efficiency is achieved on kanamycin-containing selective media when nptII is used in combination with aphA-6 rather than using aphA-6 alone [38]. ...
Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.
... A number of selectable markers are employed in attaining stable plastid transformants that includes the aadA gene (encodes aminoglycoside 3′-adenylyltransferase) that confers resistance to spectinomycin (Goldschmidt-Clermont 1991). Other selectable marker genes used in plastid transformation are nptII (Carrer et al. 1993), codA (Serino and Maliga 1997), bar (Iamtham and Day 2000), EPSPS (Ye et al. 2001), gfp (Khan and Maliga 1999), and uidA (Staub and Maliga 1995). ...
The plastid transformation is used for high level expression of certain metabolically and industrially important recombinant proteins in plants. The vector, pFaadAII, a tobacco based vector system, harbouring a chimeric gene consisting of aadA coding region from Escherichia coli with 5′ 16S rDNA promoter and 3′ untranslated transcript region (UTR) of chlamydomonas rbcL gene, located in between the intergenic regions of rp132 and trnL genes. This vector used for transformation of plastids targets the foreign sequences to the small single-copy region of the plastome. Biolistic mode of approach for chloroplast transformation in Scoparia dulcis L., was achieved by bombarding the leaf explants and spectinomycin based selection system was used for regeneration of transformed plants. Transplastomic lines have been successfully established with overall efficiency of two transgenic lines for twenty-five bombarded explants. Integration of aadA in selection based regenerants was characterized by PCR and protein accumulation analysis along with seedlings experiment obtained from selfing. The chloroplast transformation developed in this plant system will provide scope for research in plastid based metabolic engineering pathways.
... Several additional selection systems have been implemented, including use of neomycin phosphotransferase ( NPTII ) in conjunction with kanamycin [114] and a mutant anthranilate synthase α-subunit gene ( ASA2 ) conferring insensitivity to synthetic indole analogues [115] . A variety of promoters and 5' and 3' untranslated regions (UTRs) have been tested for expression levels (reviewed in [116] ). ...
Plastids are subcellular organelles which perform photosynthesis in plants and algae. They are descended from free-living photosynthetic bacteria which were engulfed by early eukaryotes ~1.5 billion years ago. Nearly all plastids retain a small genome separate from the nuclear genome of the plants in which they reside, which is termed the plastome (plastid genome). This genome encodes many of the proteins required for photosynthesis, as well as bacteria-like transcriptional and translational machinery. Biotechnological techniques for making precise modifications to the plastome have been available for three decades, nearly as long as the equivalent techniques for the plant nuclear genome. However, while nuclear genetic engineering quickly moved from a laboratory novelty to a tool used for the improvement of crop plants planted on over a billion acres, plastome engineering remains largely confined to research use. The primary reasons for this lack of application are the species restrictions and technical difficulty of the transformation process, which until recently was only possible in a few species.
In 2007, a protocol for plastid transformation was reported for Marchantia polymorpha, a thalloid liverwort classically used as a model species. Marchantia offers rapid generation time, small size, simple genetics, and asexual reproduction by means of gemmae, small disks of tissue which provide a powerful platform for live-tissue microscopy. In this thesis, tools for Marchantia plastid transformation are systematically improved by the generation of the first plastome sequence assembly for a widely used laboratory strain of Marchantia, optimisation of the transformation protocol itself, and a comparison of the in vivo activity of plastid regulatory elements through a fluorescent marker and quantitative microscopy. The highly conserved nature of the land plant plastome suggests the improvements to plastid transformation developed in Marchantia will translate to other species.
... A number of selectable markers are employed in attaining stable plastid transformants that includes the aadA gene (encodes aminoglycoside 3′-adenylyltransferase) that confers resistance to spectinomycin (Goldschmidt-Clermont 1991). Other selectable marker genes used in plastid transformation are nptII (Carrer et al. 1993), codA (Serino and Maliga 1997), bar (Iamtham and Day 2000), EPSPS (Ye et al. 2001), gfp (Khan and Maliga 1999), and uidA (Staub and Maliga 1995). ...
Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The rrn16-rrn23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKS+II as a backbone by inserting the rrn16-trnI and trnA-rrn23 sequences from Cucumis sativus L. An independent expression cassette with aadA gene encoding aminoglycoside 3′-adenylyltransferase with psbA controlling elements is added into the trnI-trnA intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon (Momordica charantia L.) was achieved by bombardment of petiole segments. The frequency of transplastomic plants yielded using standardized biolistic parameters with CuIA vector was two per 15 bombarded plates, each containing 20 petiole explants. Integration of aadA gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.
... This is likely due to the high enzymatic activity of the AadA protein and the high specificity of spectinomycin as a potent inhibitor of plastid translation. A few alternative selectable markers have been developed for tobacco plastid transformation, including the nptII gene encoding a neomycin phosphotransferase that confers resistance to kanamycin (Carrer et al. 1993), the aphA-6 gene that encodes an aminoglycoside phosphotransferase also conferring kanamycin resistance (Huang et al. 2002), and the cat gene encoding chloramphenicol acetyltransferase and conferring resistance to chloramphenicol (Li et al. 2011). However, due to their substantially lower efficiency than aadAbased selection, they have not become widely adopted. ...
Plant cells host two important organelles: mitochondria, known as the cell’s ‘powerhouse’, which act by converting oxygen and nutrients into ATP, and plastids, which perform photosynthesis. These organelles contain their own genomes that encode proteins required for gene expression and energy metabolism. Transformation technologies offer great potential for investigating all aspects of the physiology and gene expression of these organelles in vivo. In addition, organelle transformation can be a valuable tool for biotechnology and molecular plant breeding. Plastid transformation systems are well-developed for a few higher plants, however, mitochondrial transformation has so far only been reported for Saccharomyces cerevisiae and the unicellular alga Chlamydomonas reinhardtii. Development of an efficient new selection marker for plastid transformation is important for several reasons, including facilitating supertransformation of the plastid genome for metabolic engineering purposes and for producing multiple knock-outs or site-directed mutagenesis of two unlinked genes. In this work, we developed a novel selection system for Nicotiana tabacum (tobacco) chloroplast transformation with an alternative marker. The marker gene, aac(6′)-Ie/aph(2′′)-Ia, was cloned into different plastid transformation vectors and several candidate aminoglycoside antibiotics were investigated as selection agents. Generally, the efficiency of selection and the transformation efficiency with aac(6′)-Ie/aph(2′′)-Ia as selectable marker in combination with the aminoglycoside antibiotic tobramycin was similarly high as that with the standard marker gene aadA and spectinomycin selection. Furthermore, our new selection system may be useful for the development of plastid transformation for new species, including cereals, the world’s most important food crops, and could also be helpful for the establishment of a selection system for mitochondrial transformation. To date, all attempts to achieve mitochondrial transformation for higher plants have been unsuccessful. A mitochondrial transformation system for higher plants would not only provide a potential for studying mitochondrial physiology but could also provide a method to introduce cytoplasmic male sterility into crops to produce hybrid seeds. Establishing a stable mitochondrial transformation system in higher plants requires several steps including delivery of foreign DNA, stable integration of the foreign sequences into the mitochondrial genome, efficient expression of the transgene, a highly regenerable tissue culture system that allows regeneration of the transformed cells into plants, and finally, a suitable selection system to identify cells with transformed mitochondrial genomes. Among all these requirements, finding a good selection is perhaps the most important obstacle towards the development of a mitochondrial transformation system for higher plants. In this work, two selection systems were tested for mitochondrial transformation: kanamycin as a selection system in combination with the antibiotic-inactivating marker gene nptII, and sulfadiazine as a selection agent that inhibits the folic acid biosynthesis pathway residing in plant mitochondria in combination with the sul gene encoding an enzyme that is insensitive to inhibition by sulfadiazine. Nuclear transformation experiments were considered as proof of the specificity of the sulfadiazine selection system for mitochondria. We showed that an optimized sulfadiazine selection system, with the Sul protein targeted to mitochondria, is much more efficient than the previous sulfadiazine selection system, in which the Sul protein was targeted to the chloroplast. We also showed by systematic experiments that the efficiency of selection and nuclear transformation of the optimized sulfadiazine selection was higher compared to the standard kanamycin selection system. Finally, we also investigated the suitability of this selection system for nuclear transformation of the model alga Chlamydomonas reinhardtii, obtaining promising results. Although we designed several mitochondrial transformation vectors with different expression elements and integration sites in the mitochondrial genome based on the sulfadiazine system, and different tissue culture condition were also considered, we were not able to obtain mitochondrial transformation with this system. Nonetheless, establishing the sul gene as an efficient and specific selection marker for mitochondria addresses one of the major bottlenecks and may pave the way to achieve mitochondrial transformation in higher plants.
... Other successfully employed selectable marker are Kanamycin-resistance genes (neo and aphA-6, Carrer et al., 1993;Huang et al., 2002), genes conferring resistance to chloramphenicol (cat, Li et al., 2011) and 4-methylindole and 7-methyl-DL-tryptophan (asa2, Barone et al., 2009). More recently, a visual marker gene (aadA au ) that allows both selection and detection of transplastomic sectors by leaf color has been developed in tobacco (Tungsuchat- Huang et al., 2011). ...
Plants are an economic and easily scalable production system for the production of proteins of agricultural, pharmaceutical and industrial interest with minimal risk of contaminations with animal pathogens. The plastid genome represents an attractive target of genetic engineering in crop plants owed to its small size and abundant number of plastids in a single plant cell. The genetic modifications of the plastid genome, therefore, have recently emerged as an alternative for the expression of different proteins. Compared with nuclear transformation, plastid genetic engineering offers unique advantages which have stirred enormous interest among plant biotechnologists. Plastid genetic engineering is, particularly more suitable for the use of plants as biofactories. Higher plants have been proposed as an economic, safe and easily scalable production system. Several recombinant proteins have been expressed using plastid transformation, including therapeutic proteins, antibiotics, proteins with immunological properties and enzymes. In addition, plastid genetic engineering has been used for metabolic engineering of numerous pathways as well as for the expression of insecticidal toxins.
... The most commonly used selective marker gene is aadA, encoding spectinomycin resistance . Kanamycin (Carrer et al., 1993), chloramphenicol , and the amino acid analogs 4-methylindole and 7-methyl-DL-Trp (Barone et al., 2009) have also been successfully employed as selective agents. ...
... This selectable marker has been used widely in plant transformation for both the nucleus and the chloroplasts (Fraley et al., 1983;Carrer et al., 1993;Wurbs et al., 2007). It also was the first antibiotic selection system that has been reported for nuclear transformation in the green alga (Hasnain et al., 1985). ...
... This is likely due to the high enzymatic activity of the AadA protein and the high specificity of spectinomycin as a potent inhibitor of plastid translation. A few alternative selectable markers have been developed for tobacco plastid transformation, including the nptII gene encoding a neomycin phosphotransferase that confers resistance to kanamycin (Carrer et al. 1993), the aphA-6 gene that encodes an aminoglycoside phosphotransferase also conferring kanamycin resistance (Huang et al. 2002), and the cat gene encoding chloramphenicol acetyltransferase and conferring resistance to chloramphenicol (Li et al. 2011). However, due to their substantially lower efficiency than aadAbased selection, they have not become widely adopted. ...
Key message:
A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants. More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6')-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.
... Inefficient transformant selection is considered the main obstacle to the implementation of plastid transformation in rice. The most commonly used genes for transformant selection in plastid transformation are antibiotic resistance genes, such as aadA (encoding aminoglycoside 3 ′ -adenylyltransferase), nptII (encoding neomycin phosphotransferase), and CAT (encoding chloramphenicol acetyltransferase), which confer resistance to spectinomycin, kanamycin and chloramphenicol, respectively (Svab et al., 1990b;Carrer et al., 1993;Li et al., 2011). Spectinomycin is widely used as a screening substrate in plastid transformation of dicotyledonous plants due to its unique high specificity and low side-effect to plant cells. ...
D-serine is toxic to plants. D-serine ammonia lyase, which is encoded by the dsdA gene, can attenuate this toxicity with high specificity. In the present study, we explored the function of codon-optimized dsdA with tobacco plastids and rice nuclear transformation system. It was shown that dsdA gene was site-specifically integrated into the tobacco plastid genome and displayed a high level of expression. Genetic analysis of the progenies showed that dsdA gene is maternally inherited and confers sufficient D-serine resistance in tobacco. The effective screening concentrations of D-serine for seed germination, callus regeneration and foliar spray were 10, 30, and 75 mM, respectively. In addition, calluses from homozygous transgenic rice lines also showed significant tolerance to D-serine (up to 75 mM). Our study proves the feasibility of using dsdA gene as a selectable marker in both plastid and nuclear transformation systems.
... Another appealing feature of chloroplasts as platforms is that one can design operons containing intercistronic expression elements, which direct the maturation of a polycistronic transcript into independent transcription units (Lossl et al., 2005;Zhou et al., 2007;Lu et al., 2013). Selection in plants uses the aadA spectinomycin/streptomycin resistance cassette extensively, but several reports have also described successful selection using kanamycin and chloramphenicol (Carrer et al., 1993;Kumar et al., 2004;Li et al., 2011). The ability to excise the resistance cassette allows for its reuse in subsequent transformations and minimizes antibiotic use. ...
The chloroplasts found in plants and algae, and photosynthetic microorganisms such as cyanobacteria, are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals, as well as complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the production levels to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the synthetic biology tools available and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts. This article is protected by copyright. All rights reserved.
... The aadA gene is still the most commonly used selectable marker for high-frequency production of plastid transformants in many higher plant species (Table 18.1). Genes encoding resistance to other antibiotics, such as cat (chloramphenicol acetyl transferase) [116], nptII [21,106] and aphA-6 [11,20] are also possible options, and could be more appropriate in some species [104]. The amino-acid analogs 4-methylindole and 7-methyl-DL-Trp have also been successfully employed as selective agents in tobacco using the feedback-insensitive α-subunit gene (ASA2) as the selectable marker [10]. ...
For the engineering of new traits in plants and algae, modification of the plastid genome offers significant advantages. These include transgene integration by homologous recombination, high levels of expression, multigene engineering in polycistronic units, and reduced transmission through pollen. Numerous tools are available for DNA delivery, selection of transformants, removal of undesirable markers and efficient expression of single or multiple transgenes. The list of plants and algae with successful plastid transformation is increasing and includes some crop plants. Its further extension to agronomically relevant species and appropriate cultivars, together with advances in synthetic biology, will be key to the future development of plastids as green factories for the production of high-value metabolites or proteins.
In the majority of plant biotechnology laboratories throughout the world, plant transformation is a common practice to improve several traits of plants, particularly grain yield. During the experiments, only a small percentage of cells transform in the targeted population. For selection of transformed cells, it is necessary to use the selectable markers such as Neomycin phosphotransferase (nptII), Chloramphenicolacetyle transferase (Cat), Hygromycin phosphotransferase (hph), Streptomycin phosphotransferase (spt), Phosphinothrycin acetyletransferase (pat) and Dihydrofolate reductase (dhf). The majority of these and other markers detoxify different antibiotics including paromomycin, kanamycin, hygromycin, neomycin, and streptomycin etc. But as these antibiotics become resistant, most of the markers raise serious safety concerns to human health. Additionally, in case of multiple genes transfer, it also limits the availability of these selectable markers. Keeping in view the limitations of selectable markers, production of marker-free transgenic plants is becoming the global trend. Current review explored the probabilities and prospects to cope with these issues. This review describes in detail the various plant transformation systems for the development of selectable marker gene (SMG) free transgenic plants. Replacement of selectable marker with screenable marker and some worldwide examples of SMG free crop plants produced by these strategies are also discussed.
In the majority of plant biotechnology laboratories throughout the world, plant transformation is a common practice to improve several traits of plants, particularly grain yield. During the experiments, only a small percentage of cells transform in the targeted population. For selection of transformed cells, it is necessary to use the selectable markers such as Neomycin phosphotransferase (nptII), Chloramphenicolacetyle transferase (Cat), Hygromycin phosphotransferase (hph), Streptomycin phosphotransferase (spt), Phosphinothrycin acetyletransferase (pat) and Dihydrofolate reductase (dhf). The majority of these and other markers detoxify different antibiotics including paromomycin, kanamycin, hygromycin, neomycin, and streptomycin etc. But as these antibiotics become resistant, most of the markers raise serious safety concerns to human health. Additionally, in case of multiple genes transfer, it also limits the availability of these selectable markers. Keeping in view the limitations of selectable markers, production of marker-free transgenic plants is becoming the global trend. Current review explored the probabilities and prospects to cope with these issues. This review describes in detail the various plant transformation systems for the development of selectable marker gene (SMG) free transgenic plants. Replacement of selectable marker with screenable marker and some worldwide examples of SMG free crop plants produced by these strategies are also discussed.
The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.
With distribution of genetic materials and advance molecular characteristics,
the chloroplast is prokaryotic compartments within the eukaryotic plants that
have turned into a crucial source for the genetic engineering and transplastomic
plants are becoming more popular means of agricultural development with elevated
crop yield. To address global agricultural problems, genetic modification of crop
plants is a rapid and promising solution to adapt the environment-friendly and well-controlled
farming system. The transplastomic plant with high accumulation of foreign
proteins (up to 45–46% TSP) and stable transgene expression with gene
containment can be a unique choice for the agricultural innovation of coming centuries.
Although the transplastomic plants still facing encumber to ensure the full
potential exploitation and expansion as an economical means, the removal of hardness
and obstacles of this technology and commercialization can contribute for the
sustainable development of future agriculture. In this book chapter, we intend to
recapitulate the up to date development and achievement of transplastomic plant
including gene transfer procedures in plastid genomes, regulable expression of plastid
transgenes, plant trait improvement by foreign gene expression, biopharmaceuticals
production, engineering of metabolic pathways in plant, study of transformation
mediated RNA editing technologies, bio-safety issues and public concerns on transplastomic plants and overall beneficial aspects. We believe that the utilization of
transplastomic plants will ensure an eco-
The chloroplast of the unicellular green alga Chlamydomonas reinhardtii is readily amenable to molecular genetic analysis. Using particle-gun-bombardment, exogenous DNA can be introduced into the chloroplast where it will recombine into the organellar genome. Coupled with the high levels of homologous recombination this allows precisely targeted insertion of introduced DNA anywhere into this 196 kbp genome. Several different chloroplast expression vectors have been constructed in order to introduce and ectopically express foreign genes within the Chlamydomonas chloroplast. One of these genes, aphA-6, a eubacterial aminoglycoside antibiotic resistance gene, confers resistance to kanamycin and amikacin in transformed cells and so can be used as a dominant selectable marker for chloroplast transformation. Experiments to demonstrate the utility of this new marker to chloroplast molecular genetics were also carried out. The same expression vector was used in an attempt to express the protochlorophyllide oxidoreductase (POR) gene from Synechocystis sp. PCC 6803 in a chlorophyll-less Chlamydomonas double mutant (pc-ly-7) lacking active POR. In addition, the tufA gene encoding the protein elongation factor EF-Tu, from the plastid of the Apicomplexan Plasmodium falciparum was introduced into the chloroplast. This was carried out in order to attempt a functional replacement of the endogenous chloroplast EF-Tu. The results of the introduction and expression of these two foreign genes are discussed. Finally, an experiment is described which attempted to isolate cells in which chloroplast DNA had transferred to the nuclear genome. It was hoped that this would serve as a model for the process occurring throughout chloroplast evolution by which the majority of plastid genes have become nuclear encoded. To achieve this the 'ble' selectable marker for nuclear transformation in Chlamydomonas, conferring zeomycin resistance, was introduced into the chloroplast genome where it was not functional. Cells in which this marker had moved to the nuclear genome were then selected by their ability to survive on zeomycin containing medium. The results of this screen are discussed.
We designed a dicistronic plastid marker system that relies on the plastid’s ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5’ UTR of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under UV light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.
Agrobacteria virulence writ large
Plasmids are widespread among bacteria and are important because they spread virulence and antibiotic resistance traits, among others. They are horizontally transferred between strains and species, so it is difficult to work out their evolution and epidemiology. Agrobacteria, a diverse grouping of species that infect plants, inject oncogenic Ti and Ri plasmids, which cause crown galls and hairy root diseases, respectively. The upside is that these plasmids have become valuable biotechnological tools. Weisberg et al. combed through an 80-year-old collection of Agrobacterium strains but found a surprisingly low diversity of plasmids. It is puzzling how limited the number of plasmid lineages is despite reported high levels of plasmid recombination, but what is clear is how plant production systems have influenced plasmid spread into various genomic backbones.
Science , this issue p. eaba5256
Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work a complete modular chloroplast cloning system (MoChlo) was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5'UTRs, 9 promoter:5'UTR fusions, 10 3' UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes, and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to 7 transcriptional units in a single vector (~10.6 kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, will be available from Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.
Wheat is one of the most important staple food crops of the world, and continuous genetic improvement is vital to meet the demands of the rapidly growing world population. Conventional breeding has led to the development of current high yielding wheat varieties, and recent achievements in genetic engineering are expected to augment conventional breeding to further increase production. Advances in genome sequencing and molecular breeding have increased the rate of gene discovery, leading to a need for highly efficient and robust transformation systems. Targeted genome editing will require efficient delivery of sequence-specific nucleases, such as zinc fingers (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases such as CRISPR-Cas9. Since the first report of fertile transgenic wheat in 1992, optimization of plant tissue culture techniques, DNA delivery methods, gene expression cassettes, and marker genes have led to reliable transformation protocols for a range of wheat model cultivars. However, like other cereal crops, wheat transformation has also been hampered by genotype effects. The limited range of transformable tissues in wheat is considered another challenge. Several excellent review papers have described the progress made towards developing robust genetic transformation systems for wheat, so we have focused our attention on a detailed analysis of selectable markers and promoters that have been used. The choice of selectable marker and promoter can dramatically influence the outcome of a transformation project. Both Agrobacterium tumefaciens and microprojectile-mediated transformation systems have been employed successfully for genetic transformation of wheat using genes of agronomic importance. Since improvement in agronomic traits of wheat will affect a sizeable population, we have provided an analysis of the progress made towards developing genetically superior wheat containing gene(s) of agronomic importance. Recent efforts on targeted genome editing in wheat are also discussed.
Major crop losses occur annually as a result of biotic and abiotic stresses. The ability to hyperexpress foreign proteins, single-step multigene engineering, lack of positive effect and gene silencing, vector sequences and pleiotropic effects have resulted in several hundred-fold more tolerance to the environmental stresses via chloroplast genetic engineering than nuclear genetic engineering. Maternal inheritance of chloroplast expressed transgenes renders the technology environmentally safe and promotes public acceptance. This review provides protocols for engineering agronomic traits like insect, herbicide and disease resistance; salt and drought tolerance; and phytoremediation via chloroplast genome.
Major crop losses occur annually as a result of biotic and abiotic stresses. The ability to hyperexpress foreign proteins, single-step multigene engineering, lack of positive effect and gene silencing, vector sequences and pleiotropic effects have resulted in several hundred-fold more tolerance to the environmental stresses via chloroplast genetic engineering than nuclear genetic engineering. Maternal inheritance of chloro-plast expressed transgenes renders the technology environmentally safe and promotes public acceptance. This review provides protocols for engineering agronomic traits like insect, herbicide and disease resistance; salt and drought tolerance; and phyto-remediation via chloroplast genome.
Chloroplast genetic engineering offers a number of unique advantages, including high-level trans-gene expression, multigene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed via the tobacco chloro-plast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to other important plant species. For example, Arabidopsis may be an ideal model system for chloro-plast functional genomics. The employment of chloroplast transformation technology in Arabidopsis has been hampered by the lack of an efficient and reproducible protocol that provides fertile chloro-plast transgenic plants. Transformation of the Arabidopsis chloroplast genome was achieved via organogenesis but the efficiency was at least a 100-fold lower than in tobacco and had the drawback of polyploidy in the leaf tissue that resulted in sterile transgenic plants. This problem can be overcome by adapting procedures that are now available to regenerate plants from both diploid and tetraploid explants via callus. In addition, it is feasible to regenerate Arabidopsis via somatic embryogenesis. Recent breakthroughs in highly efficient plastid transformation of recalcitrant crops such as cotton and soybean have opened the possibility of engineering Arabidopsis plastid genome via somatic embryogenesis. Therefore, protocols of recent improvements in tissue culture, DNA delivery, and the novel vector designs are provided here in order to achieve highly efficient plastid transformation in Arabidopsis.
Genetic material in plants is distributed into the nucleus, plastids, and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is an advantage to nuclear gene transformation due to higher expression of transgenes, absence of gene silencing and position effect, and transgene containment by maternal inheritance, i.e., plastid gene inheritance via seed not by pollen prevents transmission of foreign DNA to wild relatives. Thus, plastid transformation is a viable alternative to conventional nuclear transformation. Many genes encoding for industrially important proteins and vaccines, as well as genes conferring important agronomic traits, have been stably integrated and expressed in the plastid genome. Despite these advances, it remains a challenge to achieve plastid transformation in non-green tissues and recalcitrant crops regenerating via somatic embryos. In this chapter, we have summarized the basic requirements of plastid genetic engineering and discuss the current status and futuristic potential of plastid transformation.
January of 1983 was a turning point for plant biotechnology when tobacco was immortalized as a surrogate biological system for testing gene function at a conference on “Advances in Gene Technology: Molecular Genetics of Plants and Animals” hosted by the Miami Winter Symposia series. Although Arabidopsis has now become the system of choice for nuclear gene integration due to the ease of transformation and a short generation cycle, tobacco remains the only established system for plastid transformation. This review summarizes the use of tobacco in dissecting plant biology concepts pertaining to the three important compartments of the cell that harbor genetic material within them. Recent studies in N. benthamiana have brought the genus back to the limelight as an outstanding system for transient protein expression. Overall, this chapter also brings out the advantages and limitations of tobacco as a system for discovery in plant biology. As a nonfood and nonfeed crop tobacco retains a remarkable potential for use as a biofactory. Ironically, this genus with a notorious health reputation may prove to be indispensable for the production of medically relevant compounds.
Biolistic transformation is the dominant method for construction of transgenic cereals. Although the biolistic approach can be optimized for transformation of any plant (and non-plant) species, the efficiency of generated transformants is significantly lower compared to Agrobacterium-based transformations. Protocols for Agrobacterium-mediated transformation of grasses have been successfully developed during the past years. However, biolistic transformation continues to be of major importance for gene technological advances in cereals and other higher plants.
The plastid genome of higher plants is a 120-kb to 160-kb double-stranded DNA present in 1,900 to 50,000 copies per leaf cell. To obtain genetically stable transplastomic lines every one of the plastid genome copies (ptDNA) should be uniformly altered in a plant. Transformation is accomplished through the following steps: (i) introduction of the transforming DNA, encoding antibiotic resistance, by the biolistic process or PEG treatment; (ii) integration of the transforming DNA by two homologous recombination events and (iii) elimination of wild-type genome copies during repeated cell divisions on a selective medium. As integration of foreign DNA always occurs by homologous recombination, plastid transformation vectors contain segments of the plastid genome to target insertions to specific locations. The plastid vectors also contain a marker for selection. Useful, non-selectable genes are cloned next to selectable marker genes, with which they are introduced into the plastid genome. Homology-directed genome manipulations have included introduction of point-mutations and deletion of targeted genes.
A new spectinomycin plus streptomycin resistant double mutant of Solanum
nigrum has base substitutions in the 16S rRNA and rps12(3′) genes on the chloroplast DNA. A cloned fragment containing both these sites was used to transform Nicotiana
plumbaginifolia protoplasts, using polyethylene glycol in a protocol previously developed for efficient chloroplast transformation. Selection of transformants was on the basis of greening of protoplast-derived colonies on antibiotic containing medium, and was for either simultaneous resistance to both antibiotics, or resistance to spectinomycin followed by screening for streptomycin resistance. Shoots have been recovered from a number of double resistant colonies and homologous integration of S. nigrum DNA confirmed by PCR using primers diagnostic for both species.
During recent years, the use of plant biotechnology and genetically modified (GM) crops has increased considerably. In 1999, transgenic soybean with engineered herbicide resistance accounted for more than 50% of the soybean grown in the US. Maize and cotton carrying an endotoxin gene from Bacillus thuringensis (Bt) exhibiting insect resistance are now widely used. A rice plant that produces enough β-carotene to supplement the daily requirements of a human and serves to enhance iron uptake was recently presented. Some of the most important milestones in the development of transgenic crops are summarized in Table 1. The data given there highlight the enormous developments that have occurred during the last two decades of this last millennium. Consequently, one may conclude that the future of GM crops during the first decade of the third millennium should be bright. However, recent years have also witnessed increasing anti-GM-crop activity. This is not only true in Europe; resistance is also growing notably overseas (Lehrman 1999), making the fate of transgenic crops somewhat uncertain. In most European countries, consumer acceptance of GM-based food products is low.
Success in bean (Phaseolus vulgaris L.) transformation requires an understanding of both tissue culture and transformations systems. Several techniques have been proposed in order to develop an efficient technology to transform bean. The susceptibility of bean to Agrobacterium has been demonstrated and electroporation or polyethylenglycol (PEG)-mediated transformation has been evaluated. Using the particle bombardment process, transgenic bean plants containing agronomically important traits from several varieties have been obtained. Some aspects influencing the achievement of transgenic P. vulgaris by particle bombardment, limitations and future prospects are discussed.
In plant transformation systems, the efficiency of stable gene transfer is low. Only a fraction of the cells exposed to the foreign DNA integrate the DNA into the genome, thus providing the basis for regeneration of transgenic plants. Therefore, systems are required which allow selection of such transformed clones. A selective system consists of a selective agent, i.e., a phytotoxic compound interfering with the plant cellular metabolism, and a selectable marker gene, i.e., a DNA sequence coding for a product which allows detoxification or evasion of the selective agent through enzymatic modification of the selective agent or expression of an altered target, respectively. Clones with integrated selectable marker genes can be detected through negative selection of untransformed cells or tissue in vitro or whole plants in vivo. Co-transformation of nonselectable sequences linked or nonlinked with selectable marker genes allows recovery of tissue or plants which contain the nonselectable sequence. A selection system for transformed plant cells must meet several requirements: the selectable marker gene should allow expression in a variety of plant tissues, the background metabolic activity or resistance should be minimal, and a clear change of phenotype should be visible. Over the past decade, a number of such systems have been developed (cf. Table 1). They include the use of antibiotics, herbicides, substrate analogues, or high concentrations of other compounds. Several of them are widely used in plant transformation work.
When Erwin Baur, at the beginning of this century, proposed that the non-Mendelian inheritance of leaf variegations can be explained with the assumption that chloroplasts (plastids) contain their own genetic information (Baur 1909, 1910), he found himself confronted with the sheer disbelief of many of his colleagues (Hagemann 1999). It took more than half a century until the discovery of chloroplast DNA (Chun et al. 1963; Sager and Ishida 1963) provided the ultimate proof for Baur’s ingenious hypothesis. Already with the very first analyses on chloroplast DNA sequences, it became obvious that plastid and eubacterial genomes are evolutionarily related (Schwarz and Kössel 1979, 1980), a finding that provided direct molecular evidence for the endosymbiotic origin of organelles (Gray 1989). The elucidation of the complete DNA sequence of two chloroplast genomes in 1986 (Ohyama et al. 1986; Shinozaki et al. 1986) marks a milestone in organelle genetics and has had a profound influence on our understanding of the biology and evolution of plastids (cf. Hagemann and Hagemann 1994; Hagemann et al. 1996, 1998).
Interest in genetic transformation of plant cells has increased the need for molecular analysis of DNA from small samples of plant tissue. The ideal method for extracting this DNA would be simple and rapid to reduce the number of steps required (and to allow a large number of extractions to be run at a time). High, reproducible yields would allow extractions from a minimum mass of tissue enabling one to assay small tissue fragments or single colonies. The ability to assay a minimum amount of tissue could save considerable time otherwise spent waiting for the treated tissue to grow to a large enough size for conventional extraction methods to be used. Finally, the DNA produced needs to be sufficiently pure to allow restriction endonuclease digestion and genomic blot analysis. The procedure outlined in this report was specifically designed to reduce the number of required steps and maintain an efficient yield of high molecular weight DNA suitable for analysis. It is derived from a combination and modification of published procedures for CsCI purification of plant DNA (Bendich et al., 1979) and miniprep of bacterial plasmids (Birboim and Doly, 1979). The basic extraction procedure consists of three stpes; 1) homogenization, 2) phenol wash, and 3) ethanol precipitation. Since the buffer volumes are minimized as well as the number of extraction steps, the DNA yield is sufficient Inquiries to." lrvin J. Mettler, Stauffer Chemical Company, 1200 So. 47th Street, Richmond, California 94804.
Transpositions of segments of R factor (antibiotic resistance plasmids) to bacteriophage lambda have been selected and characterized. Cells of Escherichia coli harboring R factors that determine kanamycin resistance were infected with phage lambda, and lambdakan transducing lines were obtained. Each of the three examined is unusual when compared to lambda transducing phages containing E. coli chromosomal genes: the kan insertions (a) occur at several sites, each well removed from the integration region POP', (b) are not associated with deletion of lambda phage DNA, and (c) are separable from the lambda genome during transduction or during lytic growth. Two insertions from the same R factor contain 1.5 kilobase sequences repeated in inverted order. The properties of the lambdakan phage suggest that R factors contain systems capable of mediating genetic exchange in the absence of extensive DNA homology. It is suggested that such systems of exchange may have played important roles in R factor evolution.
We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.
The plastid psbA promoter of tobacco was used with the aim to construct plastid specific marker genes. Upon transfer to the tobacco nuclear
genome the plastid promoter fragment appeared to specify a messenger RNA. Placed 5' to the bar or nptll coding sequences the level of expression is sufficient to obtain a selectable phenotype. The transcription start site in
the nucleus is site specific and is located 4 nucleotides downstream relative to the start site used in plastids. Translational
fusions of the psbA coding sequence with the nptll and bar coding sequences revealed that the psbA leader sequence and the psbA translation start codon, being the second ATG codon, are recognized by the plant cytoplasmic translation apparatus.
A promoter cassette utilisable in both E. coli and tobacco, was obtained by placing the CaMV 35S enhancer 5' to the psbA promoter.
The method takes advantage of a strong biological selection against the original DNA template which is preferentially destroyed on transfection. The use of this spatial template can be combined with many of the previously described in vitro mutagenesis methods. What we describe here is not, therefore, a new procedure for site-directed mutagenesis but is, rather, the use of standard and well-established procedures in conjunction with an unusual template. This combination permits flexibility in the choice of mutagenesis techniques and makes possible the highly efficient recovery of mutants.
To compare the effects of different transformation methods on the integration behavior and structural stability of integrated foreign genes in plant cells, tobacco protoplasts were transformed with Escherichia coli plasmid pLGV2103neo DNA using the Ca phosphate DNA coprecipitation technique. Parallel transformations were done by cocultivation with Agrobacterium tumefaciens harboring the Ti plasmid derivatives pGV3850::2103neo or pGV3850::1103neo. A comparison of the fine structure of the integrated donor DNA obtained by direct gene transfer and by cocultivation indicates that the donor DNA in cells transformed by the former technique undergoes structural changes and concatemerizations, while the DNA integrated by the latter procedure is often unaltered. The cotransformed nopaline synthase gene, which is present in the donor Ti plasmid DNA, was inactivated in two out of nine cases. Once integrated, the arrays of selectable marker DNA appear to be structurally stable under different cell culture and selection conditions, as well as after genetic transmission.
The plastid psbA mRNA is present in all tissues, while the encoded 32 kDa D1 protein of photosystem II accumulates tissue-specifically and in response to light. To study the regulation of D1 accumulation, a chimeric uidA gene encoding beta-glucuronidase (GUS) under control of the psbA 5'- and 3'-regulatory regions (224 and 393 bp, respectively), was integrated into the tobacco plastid genome. A high level of GUS accumulation in leaves and the lack of GUS in roots, with uidA mRNA present in both tissues, indicated tissue-specific accumulation of the chimeric gene product. Light-regulated accumulation of GUS in seedlings was shown. (i) Light-induced accumulation (100-fold) of GUS in etiolated cotyledons was accompanied by only a modest increase in mRNA levels. (ii) Inhibition of GUS synthesis was observed in cotyledons when light-grown seedlings were transferred to the dark, with no reduction in mRNA levels. Tissue-specific and light-regulated accumulation of GUS indicates that D1 accumulation is controlled via cis-acting regulatory elements in the untranslated region of the psbA mRNA. We propose that in tobacco, control of translation initiation is the primary mechanism regulating D1 protein accumulation.
Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.
The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.
Plastid engineering currently relies on DNA delivery by the biolistic process. We report here stable plastid transformation in tobacco by an alternate direct transformation protocol that is based on polyethylene glycol (PEG) treatment of leaf protoplasts in the presence of the transforming DNA. Clones with transformed plastid genomes were selected by spectinomycin resistance encoded by a mutant 16S ribosomal RNA gene. Incorporation of the transforming DNA into the plastid genome was confirmed by two unselected markers, streptomycin resistance and a novel PstI site that flank the spectinomycin resistance mutation in plasmid pZS148. Our simple and inexpensive protocol eliminates the dependence on the particle gun for chloroplast transformation and should facilitate applications of plastome engineering in crops.
The feasibility of obtaining clonal lines with replicating, multicopy geminivirus vectors by direct DNA trans-formation of cultured tobacco cells was studied. The replicating vectors pTGA32 and pST31 are based on the tomato golden mosaic virus (TGMV) A genome and encode the neomycin phosphotransferase type II (NPT-II) enzyme that confers kanamycin resistance to plant cells. Following introduction into plant cells, unit-length viral genomes were released from the tandem repeats and replicated. In protoplasts, replication of unit-length pTGA32 and pST31 was about as efficient as replication of unit-length DNA A from plasmid pTGA26, which contains 1.5 copies of wild-type DNA A. Tobacco suspension culture cells were bombarded with the recombinant DNA A constructs and selected for kanamycin resistance. The number of kanamycin-resistant clones per bombardment was about the same when the TGMV DNA A vectors or a non-replicating plasmid (pLC14) which also encodes NPT-II was used. Replicating, unit-length DNA A in up to approximately 1000 copies per cell was found in about 10% of the kanamycin-resistant clones selected following bombardment of cells with TGMV vectors. The results suggest that geminiviruses may serve as useful multicopy vectors in cultured cells.
Intermolecular recombination of Chlamydomonas chloroplast genes has been analyzed in sexual crosses and following biolistic transformation. The pattern and position of specific exchange events within 15 kb of the 22-kb inverted repeat have been mapped with respect to known restriction fragment length polymorphism markers that distinguish the chloroplast genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii. Recombinant progeny were selected from two- and three-factor crosses involving point mutations conferring herbicide (dr) and antibiotic resistance (er and spr) in the psbA, 23S and 16S ribosomal RNA genes, respectively. Exchange events were not randomly distributed over the 15-kb region, but were found to occur preferentially in a 0.7-kb sequence spanning the 3' end of the psbA gene and were much less common in an adjacent region of ca. 2.0 kb. These findings are corroborated by data showing that the dr mutation is unlinked genetically (3% recombination/kb) to the er and spr rRNA mutations, which are themselves linked and show ca. 1% recombination/kb. This discrepancy is significant since the dr-er and er-spr intervals are about the same length (ca. 7 kb). During chloroplast transformation, the 0.7-kb recombination hotspot also functions as a preferential site for exchange events leading to the integration of donor psbA gene sequences. The 0.7-kb hotspot region contains four classes of 18-37-bp direct repeats also found in other intergenic regions, but no open reading frame. Using deletion constructs in a chloroplast transformation assay, the hotspot was localized to a 500-bp region that lacks most of these repeats, which suggests that the repeats themselves are not responsible for the increased recombination frequency. Within this region, a 400-bp sequence is highly conserved between the chloroplast genomes of C. reinhardtii and C. smithii and includes several structural motifs characteristic of recombination hotspots in other systems.
Tentoxin is a naturally occurring phytotoxic peptide that causes seedling chlorosis and arrests growth in sensitive plants and algae. In vitro, it inhibits activity of the beta subunit of the plastid proton-adenosine triphosphatase (ATPase) from sensitive species. Plastid atpB genes from six closely related, tentoxin-sensitive or -resistant Nicotiana species differ at codon 83, according to their response to the toxin: glutamate correlated with resistance and aspartate correlated with sensitivity. The genetic relevance of this site was confirmed in Chlamydomonas reinhardtii by chloroplast transformation. The alga, normally tentoxin-resistant, was rendered tentoxin-sensitive by mutagenesis of its plastid atpB gene at codon 83. Codon 83 may represent a critical site on the beta subunit that does not compete with nucleotide binding or other catalytic activities.
The T-DNA region of Agrobacterium tumefaciens tumour-inducing plasmids
of the nopaline type1 contains a gene coding for the enzyme
nopaline synthase. This gene is expressed constitutively in host plant
cells to which it is transferred during tumour induction2. We
have exploited the regulatory elements of this gene to construct a
chimaeric gene that confers antibiotic resistance on transformed plant
cells. The chimaeric gene encodes the expected chimaeric transcripts in
plant cells, and confers on transformed cells the ability to grow in the
presence of normally lethal levels of the antibiotic G418 (ref. 3).
Experiments using in vitro transformation techniques on single plant
cells indicate that this antibiotic resistance can be used as a
selectable marker, and can therefore be used in selecting cells
transformed by T-DNA vectors that have had the genes for hormone
autotrophy deleted4. Plant cells transformed by such
`disarmed' T-DNA vectors can be regenerated into entire plants, whose
sexual progeny contain unaltered copies of the inciting
T-DNA5. The availability of this dominant selectable marker
should allow a wider range of experiments to be under taken using
different host plants.
A simple method for the measurement of neomycin phosphotransferase (NPT) activity in crude extracts of eukaryotic cells is described. This method is based on the elimination of interfering phosphorylated proteins by using phenol-chloroform extraction. This solution phase assay allows the detection of greater than or equal to 0.01 ng of NPT in the crude cell extract. Rapid screening of a large number of cell cultures generated in gene-transfer experiments, using NPT as a selective marker, is made possible by this simple technique. Further, the promoter strength of vector constructs used in gene therapy may also be estimated by this procedure.
Expression vectors for Chlamydomonas reinhardtii chloroplast transformation have been constructed with transcription and translation signals from chloroplast genes. The bacterial
aadA sequence, coding for aminoglycoside 3″ adenyl transferase, was inserted in these vectors and introduced into the C. reinhardtii chloroplast by particle gun transformation. The stable tranagenic expression of this foreign protein in the chloroplast confers
spectinomycin and streptomycin resistance to the transformed cells. This new marker can be used as a reporter of gene expression,
and as a portable selectable cassette for chloroplast reverse genetics. Targetted gene disruption mutants of loci required
for photosynthesis, tscA and psaC, were thus obtained. A gene disruption of an unidentified open reading frame, ORF472, remained heteroplasmic, suggesting
that it has a vital function.
The structure and functional mode of photosystem II reaction center protein D1 can be studied by analyzing the effects of amino acid substitutions within the binding niche for QB, the second stable electron acceptor of photosystem II, on herbicide binding. Here we report on site-directed mutagenesis of the psbA gene coding for the D1 protein in the unicellular alga Chlamydomonas reinhardtii. The chloroplasts of wild-type cells were transformed using the particle gun. The plasmids introduced carried an in vitro mutated fragment of the psbA gene. We obtained a double mutant with replacements of amino acids 264 and 266 and a triple mutant having an additional substitution in position 259. The sensitivities of both mutants toward several types of herbicides are given and compared with those of a mutant having only a substitution at position 264.
Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.
An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')II enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus- and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.
A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription
system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start
site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions
of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1)
are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused
complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was
examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative
terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the
16S promoter was most active when the minigene plasmid was supercoiled.
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The very high genome copy number in cytoplasmic organelles is a puzzling fact in cell biology. It is proposed here that high copy number reflects an increased need for organellar ribosomes that can only be satisfied by the increased ribosomal RNA gene number that results from genome multiplication.
Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3'-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated Mr of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).
Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.
The gene for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase/Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced amino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6-10 bp upstream from the initiation codon.
Detection of protein variants in the production of recombinant DNA products is an important and complex task. Rapid acquisition of this information permits feedback control of the production process and continuous validation of the product. Much of the technology required for rapid process monitoring is currently available or under development.
We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.
We have used the Escherichia coli beta-glucuronidase (uidA) gene as a reporter gene to localize the promoter and analyze the function of the 5' untranslated region (UTR) of the Chlamydomonas chloroplast petD gene. Using particle bombardment, petD-uidA transcriptional and translational fusion genes were introduced into the chloroplast genome in the large inverted repeat flanking the atpB gene. In transformants carrying a petD-uidA transcriptional fusion, uidA mRNA accumulated but was not translated. However, in a translational fusion that included the entire petD 5' UTR, uidA mRNA accumulated and a high level of beta-glucuronidase activity was detected. When approximately 70% of the petD 5' UTR was deleted from the translational fusion, uidA mRNA accumulation and beta-glucuronidase activity decreased 4- to 6-fold and 8-fold, respectively. Run-on transcription assays demonstrated that all strains transcribe the uidA gene at equivalent rates. Our results show that sequences essential for translation reside in the petD 5' UTR and also that sequences within the 5' UTR directly or indirectly affect mRNA stability. The expression of beta-glucuronidase under the control of chloroplast transcriptional and translational signals will facilitate further studies of chloroplast gene regulatory mechanisms.
Stable genetic transformation of the plastid genome is reported in a higher plant, Nicotiana tabacum. Plastid transformation was obtained after bombardment of leaves with tungsten particles coated with pZS148 plasmid DNA. Plasmid pZS148 (9.6 kilobases) contains a 3.7-kilobase plastid DNA fragment encoding the 16S rRNA. In the 16S rRNA-encoding DNA (rDNA) a spectinomycin resistance mutation is flanked on the 5' side by a streptomycin resistance mutation and on the 3' side by a Pst I site generated by ligating an oligonucleotide in the intergenic region. Transgenic lines were selected by spectinomycin resistance and distinguished from spontaneous mutants by the flanking, cotransformed streptomycin resistance and Pst I markers. Regenerated plants are homoplasmic for the spectinomycin resistance and the Pst I markers and heteroplasmic for the unselected streptomycin resistance trait. Transgenic plastid traits are transmitted to the seed progeny. The transgenic plastid genomes are products of a multistep process, involving DNA recombination, copy correction, and sorting out of plastid DNA copies.