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Plastid transformation in Arabidopsis thaliana
Abstract
Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance
(aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes
on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly
altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated
from the two lines were fertile.
... It required the development of efficient procedures for selection of transplastomic cell lines and workable protocols for in vitro regeneration of plants (Ruf et al., 2019). Although the feasibility of introducing foreign DNA into the Arabidopsis plastid genome was demonstrated more than 20 years ago (Sikdar, Serino, Chaudhuri, & Maliga, 1998), the generation of fertile Arabidopsis plants with stably transformed plastid genomes has become possible only recently (Ruf et al., 2019). ...
... By contrast, leaf cells of Arabidopsis exhibit a strong tendency to become polyploid (with the average ploidy level in fully expanded rosette leaves being 13C; Preuten et al., 2010). While polyploidy does not pose an obstacle to successful transformation, it leads to problems with plant regeneration, and even if plants can be regenerated, they are usually infertile (Hibberd, 2017;Sikdar et al., 1998;Yu, Lutz, & Maliga, 2017). ...
... The general feasibility of introducing foreign DNA into the Arabidopsis plastid genome was demonstrated more than 20 years ago (Sikdar et al., 1998). However, the generation of fertile transplastomic Arabidopsis plants has turned out to be challenging and has become possible only recently (Ruf et al., 2019). ...
Plastids (chloroplasts) are the defining organelles of plants and eukaryotic algae. In addition to performing photosynthesis, plastids harbor numerous other metabolic pathways and therefore are often referred to as the biosynthetic center of the plant cell. The chloroplasts of seed plants possess dozens of copies of a circular genome of ∼150 kb that contains a conserved set of 120 to 130 genes. The engineering of this genome by genetic transformation is technically challenging and currently only possible in a small number of species. In this article, we describe the methods involved in generating stable chloroplast‐transformed (transplastomic) plants in the model species Arabidopsis ( Arabidopsis thaliana ). The protocols presented here can be applied to (1) target genes in the Arabidopsis chloroplast genome by reverse genetics and (2) express reporter genes or other foreign genes of interest in plastids of Arabidopsis plants. © 2021 The Authors.
Basic Protocol 1 : Generation of root‐derived microcallus material for biolistic transformation
Basic Protocol 2 : Chloroplast transformation by biolistic bombardment of root‐derived microcalli
Basic Protocol 3 : Regeneration of transplastomic lines and seed production
... La mayoría de los trabajos consiguen plantas fértiles aunque hay otros que no lo describen. Ha habido casos en los que no se han obtenido plantas fértiles (Sikdar et al., 1998) o plantas con fenotipo normal (Skarjinskaia et al., 2003).Teniendo en cuenta todos los trabajos publicados se puede decir que, además de en tabaco, en las únicas especies en las que se han establecido protocolos de transformación plastidial lo suficientemente eficientes y reproducibles son en tomate, lechuga y soja. En el resto de especies la mayoría de los trabajos son puntuales y enfocados a la puesta a punto de la técnica (Tabla 1 a Tabla 4). ...
... Li et al. (2009) La siguiente familia con mayor número de trabajos de transformación realizados son las brasicáceas con 5 especies transformadas (Tabla 2). Los ratios de transformación en general son peores que en solanáceas: desde 1 evento/7,5 placas bombardeadas (Liu et al., 2007) a 1 transformante/95 placas bombardeadas (Sikdar et al., 1998). La aparición de escapes ha llegado a ser de un 98% (Skarjinskaia et al., 2003) y se han obtenido plantas estériles (Sikdar et al., 1998). ...
... Los ratios de transformación en general son peores que en solanáceas: desde 1 evento/7,5 placas bombardeadas (Liu et al., 2007) a 1 transformante/95 placas bombardeadas (Sikdar et al., 1998). La aparición de escapes ha llegado a ser de un 98% (Skarjinskaia et al., 2003) y se han obtenido plantas estériles (Sikdar et al., 1998). ...
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.
... Arabidopsis 69 thaliana, the most widely used model plant is one of the species that is recalcitrant to plastid 70 transformation. In Arabidopsis, only 2 transplastomic events were identified in 201 samples 71 (Sikdar et al., 1998), a sample size that would have yielded approximately 200 events in tobacco 72 using the technology available in 1998. Until now the reasons for the low efficiency in 73 Arabidopsis were not understood. ...
... 141 We obtained 8 events in 5 bombarded plates using leaf tissue in the acc2-1 mutant background 142 and 4 events in 4 bombarded plates in the Sav-0 accession (Table 1). This transformation 10 This is a significant advance, as high frequency plastid transformation in Arabidopsis has been 146 pursued since the publication of the original report in 1998 (Sikdar et al., 1998). Since 2007, 26 147 plates of RLD and 5 plates of Landsberg erecta (Ler) leaf tissue were bombarded; none of which 148 yielded a transplastomic event (Table 1). ...
... After bombardment of Col-0 and Sav-0 leaves, selection of transplastomic events was carried out according to the published RLD protocol (Sikdar et al., 1998). However, when the 180 transplastomic clones were transferred to the RLD shoot-induction medium (ASI-N1B1 181 medium), the calli did not proliferate. ...
Plastid transformation is routine in tobacco, but 100-fold less frequent in Arabidopsis, preventing its use in plastid biology. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetyl-CoA-carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-Carboxylase subunit. We bombarded ACC2-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (aadA) gene and gfp, encoding the green fluorescence protein GFP. Spectinomycin resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic mRNA, that would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin hypersensitive Slavice (Sav-0) and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Sav-0 and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.
... The first successful plastid transformation was performed in the green unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) (Boynton et al. 1988). This technique was subsequently expanded to model plants including tobacco (Nicotiana tabacum) (Svab et al. 1990) and Arabidopsis (Arabidopsis thaliana) (Ruf et al. 2019;Sikdar et al. 1998;Yu et al. 2017), as well as crop plants including potato (Solanum tuberosum) (Sidorov et al. 1999) and tomato (S. lycopersicum) (Ruf et al. 2001). ...
... In the present study, we generated multiple shoots from the model plants Arabidopsis and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) via a newly developed liquid culture system and tested their suitability as materials for fusion peptide-mediated plastid transformation. To date, shoots induced from calli have been used for genetic transformation of Arabidopsis (Sikdar et al. 1998), N. benthamiana (Davarpanah et al. 2009), and F.×ananassa (Amjad and Srivastava 2006), including their plastid transformation. Multiple shoots produced by our technique can be used to generate intact plants without the need for tissue culture or regeneration, making them suitable for simple, rapid plastid transformation in planta. ...
The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.
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... For the above reasons, plastid transformation has also proven to be a serious challenge in the model system of plant biology, Arabidopsis thaliana-a member of the mustard family (Brassicaceae). While the production of plastid-transformed Arabidopsis cells by biolistic bombardment of leaves was accomplished as early as the year 1998 22 , the regenerated plants were male and female sterile and thus could not be maintained. Recent work has made the generation of transplastomic Arabidopsis cells more efficient 23 , but has not solved the fertility problem 24 . ...
... Given that the introduction of foreign DNA into chloroplasts by biolistic transformation solely relies on physical principles and therefore occurs in a largely species-independent manner, the development of efficient protocols for the selection and regeneration of fertile transplastomic lines is key to the establishment of plastid transformation in any new species. The inefficiency of regeneration from leaves and the high incidence of (male and female) infertility of the regenerated plants has hampered the development of a transplastomic technology for Arabidopsis, even though transplastomic cells could be readily produced 22,23 . As noted previously 24 , the major "challenge is therefore to identify procedures and ecotypes that facilitate this conversion of transplastomic callus of Arabidopsis into stable and heritable plant material". ...
The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
... The chloroplast transformation was successfully established in a unicellular alga Chlamydomonas reindhartii (Boynton et al. 1988;Daniell et al. 1990) and more often in Nicotiana tabacum (Svab et al. 1990). The same technology was further extended in other plant systems like Solanum tuberosum (Sidorov et al. 1999), Brassica napus (Cheng et al. 2010), Arabidopsis thaliana (Sikdar et al. 1998), Solanum lycopersicon (Ruf et al. 2001), Glycine max (Dufourmantel et al. 2005), Brassica oleracea (Nugent et al. 2006), Lactuca sativa (Kanamoto et al. 2006), Oryza sativa , Gossypium hirsutum (Kumar et al. 2004), Lesquerella fendleri (Skarjinskaia et al. 2003) and Solanum melongena (Singh et al. 2010). ...
... Many of intergenic regions were found to be target integration sites via homologous recombination (Maliga 2004) and even though the chloroplast DNA sequences are generally highly conserved at nucleotide sequence level (Sriraman et al. 1998), use of heterologous vector in transformation that would reduce the efficiency in non specific species which may be due to lack of complete homology of targeting sequences and this could be the reason for inefficiency found when plastids of tobacco that have been transformed with Solanum nigrum (Kavanagh et al. 1999). The transformation frequency in S.dulcis using heterologous vector shows significant similarity with frequency observed in L. fendleri (Skarjinskaia et al. 2003) and obviously is more efficient than frequency observed in Arabidopsis (Sikdar et al. 1998), which was relatively low as one transplastomic clone for 100 bombarded samples. These results may suggest that similar levels of homology may not be required for the construction and using of species specific plastid transformation vectors if they are closely related, that which was conveyed from potato and tomato transformation with tobacco plastid targeting sequences. ...
In the present investigation we report stable plastid transformation in Scoparia dulcis L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring aadA as a selectable marker and egfp as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats, trnR/trnN of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.
... Chloroplast (plastid) transformation occurs via efficient gene integration through homologous recombination at a precise location. There have been reports of successful transformations in Arabidopsis (Sikdar et al. 1998), eggplant (Singh et al. 2010), rice (Wang et al. 2018), tomato (Ruf et al. 2001), carrot (Kumar et al. 2004a), lettuce (Harada et al. 2014), cabbage (Tseng et al. 2014), cotton (Kumar et al. 2004b), petunia (Avila and Day 2014), soybean (Dubald et al. 2014), and poplar (Okumura et al. 2006). This technology has been in use for the past two decades as a strategically advantageous metabolic engineering route in plants. ...
A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation experiment. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants.
... capitata [61] Brassica oleracea var. botrytis [62] Lesquerella fendleri [63] Brassica napus [64] Arabidopsis thaliana [65] Fabaceae Medicago sativa cv. Longmu 803 [66] Glycine max [67] Apiaceae Daucus carota cv. ...
Chloroplast engineering has matured considerably in recent years. It is emerging as a promising tool to address the challenges related to food security, drug production, and sustainable energy posed by an ever-growing world population. Chloroplasts have proven their potential by efficiently expressing transgenes, encapsulating recombinant proteins, and protecting them from cellular machinery, making it possible to obtain highly functional proteins. This quality has also been exploited by interfering RNA technology. In addition to the practical attributes offered by chloroplast transformation, such as the elimination of position effects, polycistronic expression, and massive protein production, the technique represents an advance in biosafety terms; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite efforts, other essential crops have remained recalcitrant to chloroplast transformation, which has limited their expansion. In this chapter, we address the most recent advances in this area and the challenges that must be solved to extend the transformation to other crops and become the de facto tool in plant biotechnology.
... Most of the activity on transformation of plant chloroplast DNA was done using the bombing method. In this way, transplastomic potatoes plants (Sidorov et al., 1999), tomatoes plants (Ruf et al., 2001), transplastomic Arabidopsis (Sikdar et al., 1998), and petunias (Zubko et al., 2004) were obtained. ...
The article is dealed with problems and prospects of experiments with biological objects directed on change of a natural hereditary basis, which include risks of creation of new unnatural living organisms that can represent biological danger to the humans and environment. We noted that the possibility of uncontrolled creation of highly pathogenic strains of microorganisms, which do not exist in nature, for humans or animals, poses a great threat. We considered that such new pathogenic strains should be dangerous to the environment and will inevitably endanger human and animal life on the planet. There are several quite convincing examples in the world illustrated the danger of invading the genetic program of a living organism, while ignoring the laws of evolution and genetics, focusing only on short-term financial benefits to individuals, companies or financial groups. We established that a key element in preventing the spread of infection from laboratories or other artificial places of consolidation is the strict implementation of standard microbiological requirements. The development of effective measures to prevent and eliminate the consequences of biological attacks, taking into account their various possible scenarios, should be based on biomedical, social, economic and legal criteria. We suggested we need to consider the cost-benefit analysis and other indices characterizing the state of ecology, economy, health, and social relations when implement the measures on biological safety. In Ukraine today there is a priority to develop a legislative position in the field of counteraction to bioterrorism. This issue concerns not only the development and adoption of special laws, but also the implementation of the policy of "efficiency" against biological threats by the relevant state authorities. This also includes the establishment of full state control over enterprises and institutions related to biological agents and toxins, and the improvement of the system of "state-scientist" to control the leakage of professional knowledge outside the country.
... Svab and Maliga (1993) successfully transformed tobacco cp (Chen et al., 2014;Yarbakht et al., 2015) using the aadA gene as a selective marker. To date, this transformation system has been applied in Arabidopsis thaliana (Sikdar et al., 1998), potato (Sidorov et al., 1999), soybean (Dufourmantel et al., 2004), rice (Lee et al., 2006), and other herbaceous plants (Liu et al., 2007;Cheng et al., 2010;Wei et al., 2011;Harada et al., 2014;Lelivelt et al., 2014;Muralikrishna et al., 2016). In the present study, the aadA gene was selected as an effective selective marker to ensure that the selected regulatory elements can perform their biological functions normally. ...
Salix floderusii is a rare alpine tree species in the Salix genus. Unfortunately, no extensive germplasm identification, molecular phylogeny, and chloroplast genomics of this plant have been conducted. We sequenced the chloroplast (cp) genome of S. floderusii for the first time using second-generation sequencing technology. The cp genome was 155,540 bp long, including a large single-copy region (LSC, 84,401 bp), a small single-copy region (SSC, 16,221 bp), and inverted repeat regions (IR, 54,918 bp). A total of 131 genes were identified, including 86 protein genes, 37 tRNA genes, and 8 rRNA genes. The S. floderusii cp genome contains 1 complement repeat, 24 forward repeats, 17 palindromic repeats, and 7 reverse repeats. Analysis of the IR borders showed that the IRa and IRb regions of S. floderusii and Salix caprea were shorter than those of Salix cinerea, which may affect plastome evolution. Furthermore, four highly variable regions were found, including the rpl22 coding region, psbM/trnD-GUC non-coding region, petA/psbJ non-coding region, and ycf1 coding region. These high variable regions can be used as candidate molecular markers and as a reference for identifying future Salix species. In addition, phylogenetic analysis indicated that the cp genome of S. floderusii is sister to Salix cupularis and belongs to the Subgenus Vetrix. Genes (Sf-trnI, Sf-PpsbA, aadA, Sf-TpsbA, Sf-trnA) obtained via cloning were inserted into the pBluescript II SK (+) to yield the cp expression vectors, which harbored the selectable marker gene aadA. The results of a spectinomycin resistance test indicated that the cp expression vector had been successfully constructed. Moreover, the aadA gene was efficiently expressed under the regulation of predicted regulatory elements. The present study provides a solid foundation for establishing subsequent S. floderusii cp transformation systems and developing strategies for the genetic improvement of S. floderusii.
... The seeds of wild-type Arabidopsis thaliana with Columbia (Col-0) ecotype background were provided by the Iranian Biological resource center (IBRC), Tehran, Iran. The seeds (25 mg) were surface sterilized treated with 70% ethanol and Hypochlorite sodium 50% as described by Sikdar et al. (Sikdar et al. 1998). The seeds were cultured on MS medium (Murashige and Skoog 1962) supplemented by sucrose 3% and agar 0.8% without phytohormones. ...
The functional diversification of the RING-finger constitutive photomorphogenesis 1 (COP1) enzyme highly depends on its (in)direct interaction with regulatory proteins involved in the Arabidopsis photomorphogenesis signaling pathways. In last decade, only a few AtCOP1-ligand complexes have been investigated using functional analysis and proved by biochemical interaction analysis, despite much more have been remaining unclear. Identifying the functions of COP1 will undoubtedly provide an opportunity for the discovery of novel COP1 targets either triggered on COP1 or targeted by COP1, which their characteristics have not been reported so far. Here, we have focused on the tertiary structure analysis of COP1 protein and characterization of its potent ligands based on the protein–protein interaction characteristics of the known AtCOP1-based protein complexes, which their interaction with COP1 were biochemically approved. Based on motif analysis and molecular docking results, a total of 88 regulatory proteins with different confidence were identified to be interactive and co-regulated with AtCOP1 E3 ligase. The correlation regulatory network analysis reveals that COP1 functions as a master switch in controlling the Arabidopsis growth and development. The up/down regulations of the potent COP1-ligands gene expression levels in cop1 knockdown T0 plants indicate the COP1 expanded roles in a wide diversity of biological processes. These characteristics consist of the photomorphogenesis signaling pathway, cell cycle regulation, cell division, histone H3-K27 methylation, stomata formation, and iron starvation. Meanwhile, the VPS11 transporter probably transports the COP1 into the endosome, lysosome, and vacuole between the nucleus and cytoplasm during the photomorphogenesis signaling pathway in darkness condition.
... Plastid transformation efficiency in Arabidopsis initially was very low (Sikdar, 1998). A breakthrough in efficiency was enabled by a 2014 study that discovered Arabidopsis accessions which were hypersensitive to spectinomycin antibiotic (Parker et al., 2014), the selective agent used to screen for transplastomic events. ...
Efficient plastid transformation in Arabidopsis (Arabidopsis thaliana) requires genetic lines that are hypersensitive to spectinomycin due to the absence of a chloroplast acetyl-coenzyme A carboxylase (ACCase) encoded in the acetyl-coenzyme A carboxylase 2 (ACC2) nuclear gene. To obtain plastid transformation-competent oilseed rape (Brassica napus), we inactivated all nuclear encoded, chloroplast targeted ACCase copies using CRISPR-Cas9. B. napus (2n = 38, AACC) is a recent interspecific hybrid of B. rapa (2n = 20, AA) and B. oleracea (2n = 18, CC) and is expected to have at least two ACC2 copies, one from each parent. The sequenced genome has two ACC2 copies, one that is B. rapa-like and one that is B. oleracea-like. We designed single guide RNAs (sgRNAs) that could simultaneously inactivate both nuclear ACC2 copies. We expressed Cas9 from a chimeric egg cell promoter 1.2 (EC1.2p) known to yield homozygous or biallelic mutants in Arabidopsis in the T1 generation. To maximize the probability of functionally inactivating both orthologs in a single step, each of the two vectors carried four sgRNAs. Four T0 transgenic lines were obtained by Agrobacterium tumefaciens-mediated hypocotyl transformation. Amplicon sequencing confirmed mutations in ACC2 genes in 10 T1 progeny, in seven of which no wild-type copy remained. The B. napus T2 seedlings lacking wild-type ACC2 gene copies exhibited a spectinomycin hypersensitive phenotype, suggesting that they will be a useful resource for chloroplast genome transformation.
... Compared to traditional methods for plastid transformation, such as biolistic methods, ptpTALECD technology has three advantages. First, it allows plastid-genome editing of A. thaliana without using specific mutants 3,4 or a specific ecotype 30 and without tissue culture, which is a major obstacle to plastid transformation. Second, it could probably be used to edit plastid genomes of other plant species that are recalcitrant to plastid transformation but amenable to nuclear transformation. ...
Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells1. We applied this system to targeted base editing in the Arabidopsis thaliana plastid genome. The targeted Cs were homoplasmically substituted to Ts in some plantlets of the T1 generation and the mutations were inherited by their offspring independently of their nuclear-introduced vectors.
... It is essential for the regeneration strategy of transplastomic plants to identify transplastomic events. It took 5 to 12 weeks for the RLD (reduced number of long days) ecotype to regenerate in 1998 (Sikdar et al., 1998). The use of spectinomycin-sensitive knockout lines acc2 and the dicistronic operon vector pATV1 shortened the period for transplastomic event detection to 3 to 5 weeks. ...
In contrast with traditional nuclear gene transformation, transplastomic technology has opened a new horizon for the transgenic plant research that offers several beneficial aspects including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. However, this technology has been well adopted and established in tobacco, the introduction and adoption of cost-effective, swift, and reproducible protocol for in vitro regeneration of transplastomic potato is challenging and laborious. The present research aimed to develop such prompt and efficient protocol to instigate and revive the regeneration potential with the combinations of different plant growth regulators (PGRs). Leaves and internodal explants from four potato cultivars were transformed with chloroplast transformation vector via particle bombardment and cultured on MS media supplemented with suitable PGRs and selection agents. Leaf explants of cultivar Kuroda induced highest (92%) number of calli where cultivar Sante produced the highest (85.7%) transplastomic shoots. Thidiazuron was found more proficient (41%) for shoot regeneration. Finally, within only seven weeks, we got 21 spectinomycin resistant shoot, and 16 of those showed integration of target genes into the plastome in PCR screening.
... Later, the aadA gene was used by Svab and Maliga [35] to transform tobacco plants, obtaining up to a 100fold frequency of transformation. These results were so satisfactory that the aadA gene is still used today for the transformation of various plant species e.g., Arabidopsis [36], tomato [37], oilseed rape [38], carrot [39], petunia [40], soybean [41], cauliflower [42], and bitter melon [43] (Table 1). ...
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
... However, plastid transformation was achieved in sugarcane, but low transformation efficiency and heteroplasmy remained to be resolved. The transformation efficiency resulting in one transformation event per 27 bombarded plates was found to be lower than reported in Arabidopsis [25,26], potato [27][28][29], and tomato [30,31]. However, transformation efficiency was comparable with rice [11] since target tissues in both cases were embryogenic calli. ...
We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARES (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll, cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.
... Boynton, Gillham, and coworkers (Boynton et al., 1988) were succeeded in plastid transformation in a unicellular alga, Chlamydomonas reinhardtii, in 1988 followed by transformation of the plastid genome in 1990 in tobacco (Svab et al, 1990) and further reached out to numerous other higher plants, such as Arabidopsis (Sikdar et al., 1998), potato (Sidorov et al., 1999), tomato (Ruf et al., 2001), rape (Hou et al., 2003), ...
For many reasons tobacco is extensively used as a model plant in transformation research and used for this study as well. Tobacco plants are cultivated in several countries on large scale. Tobacco has numerous essential traditional and modern uses in clinical field. The principle aim of this present study was to optimize the conditions for seed germination, best shooting regeneration media, optimization of antibiotics spectinomycin and kanamycin for wild type plants and in addition, to develop a protocol for efficient genetic transformation of tobacco plant cv. Petit Havana with Agrobacterium tumefaciens. Seeds of tobacco were germinated on different media without any plant growth regulators. 100% seeds germination efficiency was observed on ½ MS and best sterilization time of tobacco with ethanol was for 1 minute which also showed 100% germination efficiency. RMOP (NAA and BAP) media showed the highest shoots regeneration efficiency for both explants nodes and leaves which is nodes showed 75% and leaves showed 55% regeneration efficiency among the different media which is used for regeneration. 150 mg/L concentration of kanamycin and 500 mg/L concentration of spectinomycin for both explants (nodes and leaves) was optimized for the selection of transgenic tissues. For transformation infection time for 5 minutes and co-cultivation time period of two days showed maximum transformation efficiency of 83.33% as compared to other methods. Histochemical GUS (β-glucuronidase) assay was used to check the GUS gene expression in the nodal explants. GUS gene primers were used which amplified a 708bp fragment which also confirmed the transformation of GUS gene by Agrobacterium tumifaciens strain C58C1 containing vector p35SGUSINT. Taken together, the present study develops a tissue culture protocol and basis for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Petit Havana using nodal explants.
Keywords: Tobacco, Spectinomycin, Kanamycin, Agrobacterium tumefaciens, GUS, Infection time, Co-cultivation time.
... 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.
... On the other hand, it has been difficult to generate even heteroplasmic plastid transformants in other plant species. In Arabidopsis and rice, selection using aminoglycoside 3′-adenylyltransferase (aadA) does not work as sufficiently as it does in tobacco, resulting in the production of heteroplasmic T 0 plants only (Lee et al. 2006;Sikdar et al. 1998). ...
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.
... First successful plastid transformation was reported in Chlamydomonas reindhartii (Boynton et al. 1988). Later, it has been expanded to various higher plants i.etobacco (Svab et al. 1990), potato (Sidorov et al. 1999a), Brassica (Cheng et al. 2010), Arabidopsis (Sikdar et al. 1998), tomato (Ruf et al. 2001), Soybean (Dufourmantel et al. 2004), lettuce (Kanamoto et al. 2006), Oryza sativa (Lee et al. 2006;Wang et al. 2018), cotton (Kumar et al. 2004b), brinjal (Singh et al. 2010), Scoparia Narra et al. 2018a), Maize (Sidorov et al. 2019) and Capsicum (Kota et al. 2019),etc. Flanking regions of insertional sequences and selectable markers are the crucial elements to develop species-specific chloroplast transformation vector (Wang et al. 2009). ...
The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.
Keywords : Scoparia dulcis L., Heterologous vectors, CaIA plastid vector, Inverted repeat region, Transplastomic lines
... It has been established in several other species, including Haematococcus pluvialis [10], Dunaliella tertiolecta [11], Cyanidioschyzon merolae [12] and Phaeodactylum tricornutum [13]. Although chloroplast transformation has been reported in several higher plants species [14][15][16][17][18], the transformation of algal chloroplast offers certain advantages over higher plants. For example, the higher plants contain several thousand copies of the chloroplast genome in each cell, the transformation of each of those is quite challenging [19]. ...
In recent years, microalgae have emerged as an alternative platform for large-scale production of recombinant proteins for different commercial applications. As a production platform, it has several advantages, including rapid growth, easily scale up and ability to grow with or without the external carbon source. Genetic transformation of several species has been established. Of these, Chlamydomonas reinhardtii has become significantly attractive for its potential to express foreign proteins inexpensively. All its three genomes – nuclear, mitochondrial and chloroplastic – have been sequenced. As a result, a wealth of information about its genetic machinery, protein expression mechanism (transcription, translation and post-translational modifications) is available. Over the years, various molecular tools have been developed for the manipulation of all these genomes. Various studies show that the transformation of the chloroplast genome has several advantages over nuclear transformation from the biopharming point of view. According to a recent survey, over 100 recombinant proteins have been expressed in algal chloroplasts. However, the expression levels achieved in the algal chloroplast genome are generally lower compared to the chloroplasts of higher plants. Work is therefore needed to make the algal chloroplast transformation commercially competitive. In this review, we discuss some examples from the algal research, which could play their role in making algal chloroplast commercially successful.
... Although chloroplast transformation has been reported in several higher plant species too [25][26][27][28][29][30], the transformation of algal chloroplast offers certain advantages over higher plants. For example, higher plants contain several thousand copies of the chloroplast genome in each cell, transformation of each of those is quite challenging [31]. ...
The last few years have seen an ever‐increasing interest in the exploitation of microalgae as alternative platform to produce high‐value products, including biofuels, industrial enzymes, therapeutic proteins, including antibodies, hormones, and vaccines. Due to some unique attractive features, engineering of the chloroplast genome provides a promising platform for the production of high value targets because it allows manipulation of metabolic processes in ways that would be impossible, or at least prohibitively difficult through traditional approaches. Since its initial demonstration in 1988 in Chlamydomonas reinhardtii, genetic tools have been developed which have made it possible to produce high‐value molecules in different species. However, the commercial application of microalgae as production platform is hindered by many factors like poor biomass, low product yields and costly downstream processing methodologies. In this review, we discuss the potential of microalgae to use as an alternative production platform for high value targets using chloroplast transformation technology. This article is protected by copyright. All rights reserved
... A notable exception is that studies involving plastid genome engineering are carried out in tobacco (Nicotiana tabacum), the only vascular plant species in which plastome engineering is routine (Bock, 2015). Plastid transformation in Arabidopsis was reported in 1998, but only one transplastomic event was obtained per 100 bombarded samples, an efficiency 100 times lower than that in tobacco (Sikdar et al., 1998). A clue to why Arabidopsis plastid transformation was inefficient came years later from a study on nuclear genes essential for survival in the absence of chloroplast translation. ...
New transformation-competent Arabidopsis lines, with new plastid transformation vectors and a protocol for measuring plastid transformation efficiency will advance the engineering of the plastid genome in Arabidopsis.
... In the present investigation, we explained the cloning of inverted repeated regions, trnI/trnA from C. sativus and construction of a pKS + -based chloroplast transformation vector: CuIA containing aadA expression cassette with tobacco plastid regulatory elements. Similar consecutive studies for transformation of foreign gene cassette controlled by tobacco plastid regulatory elements were also used in the (Sikdar et al. 1998;Ruf et al. 2001;Skarjinskaia et al. 2003). Based on our experimental data and other available examples for plastid transformation, it seems likely that a species-specific promoter is not always required for construction of selection marker cassette in plastid vector development. ...
... To address these problems research was focused on the use of selectable markers, the manipulation of gene expression-control elements and the development of efficient tissue-culture systems [9]. Nevertheless, the inefficient incorporation of foreign DNA into plastomes, such as occurs in Arabidopsis thaliana [10], remains to be overcome. However, incorporating transgenes into the plastome is only the first step in obtaining a genetically stable transgenic plant because several cell generations are necessary to dilute out all wild-type plastome copies [8]. ...
Plastid transformation offers the unique advantages of high-level transgene expression and increased trans-gene containment compared with conventional trans-genic technologies. The process relies on the homologous recombination machinery of the plastid incorporating foreign DNA into the plastome, which restricts the method to species where this type of incorporation works well. However, Pal Maliga and colleagues have recently reported a novel approach for integrating foreign DNA into the plastid genome that works independently of homologous recombination. The transgenic plastid system Plastid transformation is accomplished using a multistep process in which the transformation vectors contain a selectable marker gene and passenger gene(s) flanked by plastid targeting sequences [1]. Targeting sequences enable foreign genes to be incorporated into the predetermined location through two crossover events within the homologous flanking regions. Insertion of transgenes in intergenic regions without interference with flanking genes has been successful at 14 sites throughout the plastome [2], either by biolistic DNA delivery or by polyethylene glycol treatment. Nevertheless, interference was found at one site and knockouts have been reported for 28 plastid genes. Plastid genetic engineering is emerging as an alternative new technology in plant biotechnology. Compared with conventional transgenic technologies, plastid engineering offers several potential advantages that have been extensively reviewed [1-3]. Plastid transformation has mostly been confined to Nicotiana tabacum [3] and it has taken almost ten years to extend the technology to other plant species, including Solanum tuberosum [4] and Lycopersicon esculentum [5]. Plastid transformation has also been reported for other plants including Brassica napus [6], Lesquerella fendleri [7] and Oryza sativa [8]-although problems with heteroplasmy and plant fertility have yet to be resolved. To address these problems research was focused on the use of selectable markers, the manipulation of gene expression-control elements and the development of efficient tissue-culture systems [9]. Nevertheless, the inefficient incorporation of foreign DNA into plastomes, such as occurs in Arabidopsis thaliana [10], remains to be overcome. However, incorporating transgenes into the plastome is only the first step in obtaining a genetically stable transgenic plant because several cell generations are necessary to dilute out all wild-type plastome copies [8]. Pal Maliga and colleagues recently outlined an alternative approach to incorporate foreign DNA into the plastome efficiently. The technique uses the phiC31 Streptomyces phage site-specific inte-grase (INT) to catalyze recombination to yield stable transplastomic plants [11]. Integrase-mediated recombination system Site-specific recombination systems are common in pro-karyotes and lower eukaryotes. They participate in integrating bacteriophages into host genomes. In these systems, recombinations are catalyzed by phage-encoded recombinases. These recombinases can be grouped into two types based on limited amino acid sequence homology and catalytic residues (either serine or tyrosine). The serine recombinases are referred to as the invertase or resolvase and posses a catalytic serine residue, whereas the tyrosine group members are referred to as l integrases and use a tyrosine residue during DNA cleavage. The phiC31 Streptomyces phage-encoded integrase belongs to the serine family of recombinases. Like other family members, the phiC31 phage integrase brings two recom-bination sites (the bacterial attB and phage attachment attP sites) together in a synapse and then catalyzes a concerted four-strand staggered break in the DNA sub-strates while forming transient covalent attachments with the recessed 5 0 ends [12]. One pair of half sites rotates by 1808 relative to the other to form the recombinant configuration, followed by ligation of the DNA backbone. Thus, the integration reaction generates recombinant junctions attL and attR as products. During integration, the attB and attP sites share only 3 bp sequence identity at the crossover point and 16 bp within a span of w50 bp. The crossover point for both the attachment sites is flanked by different inverted repeats (in the region of 34 bp of attB and 39 bp of attP sites) that perhaps provide binding sites for the integrase [12]. It has been observed that some integrases function autonomously whereas others catalyze the reaction with the help of DNA-binding proteins, termed accessory factors (recombination directionality factors, RDFs [13]), to influence the choice of substrates that are recombined by their cognate recombinase. The best-studied RDF is Xis, encoded by phage l, which is required in the excision reaction between attL and attR junctions to generate attP and attB as products but inhibits integrative Corresponding author: Khan, M.S. (sarwarkhan_40@hotmail.com).
... Brassica napus is not only an important agricultural crop plant, but transplastomic B. napus lines could also serve as a platform to introduce transplastomic plastids into A. thaliana. So far, the model plant A. thaliana could not be used in studies about fundamental aspects of the transplastomic system, because it does not give rise to fertile plastid transformants (Sikdar et al. 1998). Somatic hybrids between B. napus line T36 and Arabdidopis could be produced via protoplast fusion (Forsberg et al. 1998) and selected via spectinomycin resistance and/or glufosinate resistance. ...
Plastid-encoded genes are maternally inherited in most plant species. Transgenes located
on the plastid genome are thus within a natural confinement system, preventing their distribution via pollen. However, a low-frequency leakage of plastids via pollen seems to be universal in plants. Here we report that a very low-level paternal inheritance in Arabidopsis thaliana occurs under field conditions. As pollen donor an Arabidopsis accession (Ler-Ely) was used, which carried a plastid-localized atrazine resistance due to a point mutation in the psbA gene. The frequency of pollen transmission into F1 plants, based on their ability to express the atrazine resistance was 1.9 9 10-5. We extended our analysis to another cruciferous species, the world-wide cultivated crop Brassica napus. First, we isolated a fertile and stable plastid transformant (T36) in a commercial cultivar of B. napus (cv Drakkar). In T36 the aadA and the bar genes were integrated in the inverted repeat region of the B. napus plastid DNA following particle bombardment of hypocotyl segments. Southern blot analysis confirmed transgene integration and homoplasmy of
plastid DNA. Line T36 expressed Basta resistance from the inserted bar gene and this trait was used to estimate the frequency of pollen transmission into F1 plants.Afrequency of\2.6 9 10-5 was determined in the greenhouse. Taken together, our data show a very low rate of paternal plastid transmission in Brassicacea. Moreover, the establishment of plastid transformation in B. napus facilitates a safe use of this important crop plant for plant biotechnology.
... In the present investigation, we explained the cloning of inverted repeated regions, trnI/trnA from C. sativus and construction of a pKS + -based chloroplast transformation vector: CuIA containing aadA expression cassette with tobacco plastid regulatory elements. Similar consecutive studies for transformation of foreign gene cassette controlled by tobacco plastid regulatory elements were also used in the (Sikdar et al. 1998;Ruf et al. 2001;Skarjinskaia et al. 2003). Based on our experimental data and other available examples for plastid transformation, it seems likely that a species-specific promoter is not always required for construction of selection marker cassette in plastid vector development. ...
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.
... 8 Recently, a plastid transformation system in the Arabidopsis thaliana (At) Slavice (Sav-0) accession, which is spectinomycin hypersensitive, was reported and established. 9 Although plastid transformation has been reported in the model plant At Columbia (Col-0) accession, 10 such work is not routinely performed. ...
Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms, including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.
... In the present investigation, we explained the cloning of inverted repeated regions, trnI/trnA from C. sativus and construction of a pKS + -based chloroplast transformation vector: CuIA containing aadA expression cassette with tobacco plastid regulatory elements. Similar consecutive studies for transformation of foreign gene cassette controlled by tobacco plastid regulatory elements were also used in the transformation of Arabidopsis, Tomato and L. fendleri (Sikdar et al. 1998;Ruf et al. 2001;Skarjinskaia et al. 2003). Based on our experimental data and other available examples for plastid transformation, it seems likely that a species-specific promoter is not always required for construction of selection marker cassette in plastid vector development. ...
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.
... False positive regenerates have been previously reported in Arabidopsis, potato and tomato (Svab und Maliga 1993, Sikdar et al. 1998, Sidorov 1999, Ruf et al. 2001. To solve this problem of false positives, a double selection pressure of transformed plants on spectinomycin and streptomycin has been exerted (Koop et al. 1996, Kavanagh et al. 1999. ...
Chloroplast transformation has gained strong interest in regard to production of vaccines in plants. Numerous advantages are pledging for the expression of vaccine antigens through the chloroplast. Environmental friendliness, the possibility of multiple transgene expression, precision of transgene integration and lack of epigenetic effects are the main advantages provided by plastid transformation. Due to economic limitations in the availability of vaccines against infectious diseases, it is necessary to develop improved vaccines which are more efficient and cost effective and hence, easily affordable to most patients in resource-poor countries.
... Natural variation for plastome structure should be tested across more Orobanchaceae species, both to acquire better ideas of the lineages and rates of such rearrangements, but also to provide the raw material for comparisons of the functional outcomes of these rearrangements. With the ability to transform chloroplasts now available in many plant species [80][81][82][83], investigations of natural plastome variation and its role in organismal function could be directly compared to experiments that test these gene effects in engineered transgenics. ...
Background
The chloroplast genomes (plastome) of most plants are highly conserved in structure, gene content, and gene order. Parasitic plants, including those that are fully photosynthetic, often contain plastome rearrangements. These most notably include gene deletions that result in a smaller plastome size. The nature of gene loss and genome structural rearrangement has been investigated in several parasitic plants, but their timing and contributions to the adaptation of these parasites requires further investigation, especially among the under-studied hemi-parasites.
Results
De novo sequencing, assembly and annotation of the chloroplast genomes of five photosynthetic parasites from the family Orobanchaceae were employed to investigate plastome dynamics. Four had major structural rearrangements, including gene duplications and gene losses, that differentiated the taxa. The facultative parasite Aureolaria virginica had the most similar genome content to its close non-parasitic relative, Lindenbergia philippensis, with similar genome size and organization, and no differences in gene content. In contrast, the facultative parasite Buchnera americana and three obligate parasites in the genus Striga all had enlargements of their plastomes, primarily caused by expansion within the large inverted repeats (IRs) that are a standard plastome feature. Some of these IR increases were shared by multiple investigated species, but others were unique to particular lineages. Gene deletions and pseudogenization were also both shared and lineage-specific, with particularly frequent and independent loss of the ndh genes involved in electron recycling.
Conclusions
Five new plastid genomes were fully assembled and compared. The results indicate that plastome instability is common in parasitic plants, even those that retain the need to perform essential plastid functions like photosynthesis. Gene losses were slow and not identical across taxa, suggesting that different lineages had different uses or needs for some of their plastome gene content, including genes involved in some aspects of photosynthesis. Recent repeat region extensions, some unique to terminal species branches, were observed after the divergence of the Buchnera/Striga clade, suggesting that this otherwise rare event has some special value in this lineage.
... Because of these advantages, the plastid genome DNA (ptDNA) of higher plants is an attractive target for biotechnology applications. Positive plastid gene transformation has been carried out in tobacco (Svab et al., 1990), Arabidopsis (Sikdar et al., 1998), potato (Sidorov et al., 1999), tomato (Ruf et al., 2001), carrot (Kumar et al., 2004) and rice (Khan and Maliga (1999). However, plastid transformation is routine only in tobacco as its transformation efficiency is much higher than in other plants (Maliga, 2004). ...
Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA-MRS and CinH-RS2, to precisely excise a marker gene from the plastid genome of tobacco. Transplastomic plants transformed with the pTCH-MRS and pTCH-RS2 vectors, containing the visual reporter gene DsRed flanked by directly oriented MRS and RS2 recognition sites, respectively, were crossed with nuclear-genome transformed tobacco plants expressing plastid-targeted ParA and CinH recombinases, respectively. One hundred percent of both types of F1 hybrids exhibited excision of the DsRed marker gene. PCR and Southern blot analyses of DNA from F2 plants showed that approximately 30% (CinH-RS2) or 40% (ParA-MRS) had lost the recombinase genes by segregation. The post-excision transformed plastid genomes were stable and the excision events heritable. The ParA-MRS and CinH-RS2 recombination systems will be useful tools for site-specific manipulation of the plastid genome and for generating marker-free plants, an essential step for reuse of SMG and for addressing concerns about the presence of antibiotic resistance genes in transgenic plants. This article is protected by copyright. All rights reserved.
... The chloroplast transformation vectors utilize homologous flanking sequences for recombination and insertion of foreign genes. Transformation of Arabidopsis, potato, and tomato chloroplast genomes was achieved via organogenesis but the efficiency was much lower than tobacco (Sikdar et al., 1998;Sidorov et al., 1999;Ruf et al., 2001). In Arabidopsis, one chloroplast transgenic line per 40 or 151 bombarded plates was obtained but they were not fertile. ...
The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins have been demonstrated by in vitro assays and animal studies. Oral delivery of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrots) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.
... Besides the low efficiency, only sterile chloroplast transgenic plants were obtained in Arabidopsis. The authors cited extensive polyploidy of the leaf tissue as the reason for obtaining sterile plants ( 33). This problem could be overcome by adapting procedures that are now available to obtain regenerants from both diploid and tetraploid explants. ...
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.
... The mean abundance value of FEPZ is 6.5 and that of nonindustrial site is 7.4. Variation in Lepidopteran diversity was investigated in northern Borneo under different disturbance regimes and habitat gradients [45,46,47,48] Establishment of FEPZ in Falta resulted in major change in landscape pattern. The depleted abundance value in FEPZ may be due to habitat destruction in and around Falta. ...
Six arthropod orders, viz., Hemiptera, Orthoptera, Hymenoptera, Lepidoptera, Coleoptera and Araneae and their associations with plant species in the Falta Export Processing Zone (FEPZ) and non-industrial area West Bengal, India, is addressed. The study was conducted in fifteen study sites of which ten are within the industrial area covering 2-10 km of aerial distance of FEPZ and five sites of non-industrial area are 20 km to 32 km away of the industrial complex. A total of 139 species of arthropods are recorded covering 5 insect orders and one arachnid order Araneae which include 121 genera, 45 families and 5 sub-families of the above mentioned orders. This arthropod community were collected from 74 species of plants belonging to 30 families. These plants are commonly distributed in all the 15 study sites. Plant families, Asclepiadaceae, Poaceae, Euphorbiaceae and Solanaceae harbour species of five arthropod orders. Caparidaceae and Cypreraceae harbour only orthopteran insects while Polygonaceae and Boraginaceae are infested by hymenopteran insects, and Chenopodiaceae and Caesalpiniaceae harbour only hemipteran insects. Plant families, Anonaceae, Saporaceae, Acanthaceae, Nyctaginaceae, Verbenaceae, Dioscoriaceae, Plamnaceae and Flacourtiaceae harbour only coleopteran insects. Two families, Amaranthaceae and Rubiaceae harbour both coleopteran insects and arachnids whereas plants of Rhamnaceae family harbour only arachnids. The study confers comparative distribution of insects among plant species depending on their trophic relationship while Araneae maintains a housing affiliation with the inhabiting plants.
... This method of chloroplast recombination has proven to be more efficient than that OPEN ACCESS of nuclear gene transformation due to great potential, high protein levels, the feasibility of expressing multiple proteins from polycistronicm RNAs, gene containment through the lack of pollen transmission and pleiotropic effects due to sub-cellular compartmentalization of transgene products [4,8,9,10]. Positive gene transformation in chloroplast has been carried out in plants like tobacco (Nicotiana rustica) and (Nicotiana tabacum) [11,7], Arabidopsis (Arabidopsis thaliana) [12], rice (Oryza sativum) [13] potato (Solanum tuberosum) [14], carrot (Daucus carota), and tomato(Solanum lycopersicum) [15,16]. However, plastid transformation is mainly restricted to tobacco as its efficiency is much higher than in other plants [16]. ...
Chloroplasts are the site of photosynthesis in plants mostly seen in leaves and some eukaryotic algae that provides the primary sources of the world's food productivity. Plastids of higher plants are generally semiautonomous with a ~120–150 kb genome. Chloroplast transformation has become an attractive alternative to nuclear gene transformation due to its advantages, high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. The review presents the recent trends and methods for plastid genome engineering and transgene expression and summarizes the potential of plastid transformation in various fields of biotechnology and also as a source of therapeutic proteins.
... The development of transformation technologies to engineer the chloroplast genome of the green alga (Chlamydomonas reinhardtii) (Boynton et al., 1988) and Nicotina tabacum (Svab et al., 1990) opened the way to contain transgenes in the chloroplast genome (Ruf et al., 2001). The use of chloroplast transformation (chloroplast genetic engineering) in higher plants is now routinely available only for Nicotina tabacum, although some progress has been made with Arabidopsis (Sikdar et al., 1998), potato (Sidorov et al., 1999), and tomato (Ruf et al., 2001) chloroplast transformation. Such plants are called transplastomic GM lines. ...
... Later on, the first stable plastid transformation was carried out in tobacco [152]. The plastid transformation has since been established in many other plants such as Arabidopsis [153], tomato [147], potato [154], lettuce [155], carrot [156], eggplant [157], cabbage [158], sugar beet [159], alfalfa [160], rape [161], soybean [162], poplar [163], cauliflower [164], cotton [165] and petunia [166]. Furthermore, chloroplasts of nonvascular plants, like the bryophytes Physcomitrella patens [167] and Marchantia polymorpha [168] have also been transformed. ...
Background:
Vaccines produced in plants have opened up new opportunities in vaccination.
Objective:
Among the various categories of vaccines, the recombinant vaccine is generally regarded as the most economical and safest type because it cannot cause disease and does not require large-scale cultivation of pathogens. Due to the low cost of their cultivation, plants may represent viable alternative platforms for producing subunit vaccines. Genetic engineering of plastids is the innovation of the last three decades and has numerous benefits when compared to nuclear transformation. Due to the high level of expression, oral vaccines produced in transplastomic plants do not have to be purified as they can be consumed raw, which, therefore, reduces the cost of preparation, transportation and handling of the vaccines. Oral vaccination also excludes the risk of other infections or contaminations, while compartmentation of the plant cell provides an excellent encapsulation to the antigen within the plastid.
Results & conclusion:
Herein we review the main biotechnological and immunological aspects of the progress achieved in the field of plastid derived edible vaccines during the last decade. As there is a public debate against genetically modified crops, the advantages and limitations of oral vaccines are also discussed.
Chloroplast transformation has emerged as a promising platform for the development of modified transplastomic plants expressing many useful products including pharmaceutical drugs, enzymes, biomaterials, and products related to the agriculture industry. Due to high copy number of chloroplasts in green plants, it is an attractive technology for the high mass production of expressed foreign proteins. Moreover, multiple genes can be expressed into plants in a single transformation event without any epigenetic effects because of operon system of chloroplasts like prokaryotes. Plastid transformations solve the problem of expressed transgene containment, gene silencing, and minimum pleiotropic effects, which are predominant in nuclear transformation. In this chapter, we have discussed the benefits of chloroplast transformation in plant molecular farming, different methods used for plastid transformation, application of chloroplast bioengineering in difference fields of science, and advantages and limitations of chloroplast transformation for plant molecular farming.
The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0 mg/l), NAA (0.2 mg/l), TDZ (2.0 mg/l) and GA3 (0.1 mg/l); spectinomycin 50 mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.
Chloroplast is a discrete, highly structured, and semi-autonomous cellular organelle. The small genome of chloroplast makes it an up-and-coming platform for synthetic biology. As a special means of synthetic biology, chloroplast genetic engineering shows excellent potential in reconstructing various sophisticated metabolic pathways within the plants for specific purposes, such as improving crop photosynthetic capacity, enhancing plant stress resistance, and synthesizing new drugs and vaccines. However, many plant species exhibit limited efficiency or inability in chloroplast genetic transformation. Hence, new transformation technologies and tools are being constantly developed. In order to further expand and facilitate the application of chloroplast genetic engineering, this review summarizes the new technologies in chloroplast genetic transformation in recent years and discusses the choice of appropriate synthetic biological elements for the construction of efficient chloroplast transformation vectors.
The plastid (chloroplast) genome of higher plants is an appealing target for metabolic engineering via genetic transformation. Although the bacterial-type plastid genome is small compared with the nuclear genome, it can accommodate large quantities of foreign genes that precisely integrate through homologous recombination. Engineering complex metabolic pathways in plants often requires simultaneous and concerted expression of multiple transgenes, the possibility of stacking several transgenes in synthetic operons makes the transplastomic approach amazing. The potential for extraordinarily high-level transgene expression, absence of epigenetic gene silencing and transgene containment due to the exclusion of plastids from pollen transmission in most angiosperm species further add to the attractiveness of plastid transformation technology. This minireview describes recent advances in expanding the toolboxes for plastid genome engineering, and highlights selected high-value metabolites produced using transplastomic plants, including artemisinin, astaxanthin and paclitaxel.
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-
An efficient route to the transformation of Arabidopsis plastids will allow the full power of molecular genetics to be brought to bear on the study of this key compartment.
Although organelle (mitochondria and plastid) genomes have less than 1% of the genes in the nucleus, they encode essential genes, such as those involved in energy production, respiration, and photosynthesis, and genes that control agronomically important characteristics such as cytoplasmic male sterility. Organelle genomes have high copy numbers in each cell (one to two orders of magnitude greater than in the nucleus) and are characterized by maternal inheritance. To know functions of genes encoded in the organelle genomes or to develop new plants adapted to various severe environments, genetic engineering of organelle genomes is one of the promising approaches. However, modifying the mitochondrial or plastid genomes in rice is presently impossible or difficult. Here, we discuss the characteristic features of these genomes and recent attempts at plastid transformation.
The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.
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.
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.
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 chloroplast genes coding for the 16S ribosomal RNA from several spectinomycin-resistant Nicotiana mutants were analyzed. Two classes of mutants were identified. In one class, a G to A base transition is found at position 1140 of the tobacco-chloroplast 16S rRNA gene, which eliminates an AatII restriction endonuclease site. This base transition is proximal to a mutation previously described for spectinomycin resistance in Escherichia coli. In the other class, a novel G to A transition is found at position 1012 of the 16S rRNA gene. Although the mutations in the two classes are 128 nucleotides apart, the secondary structure model for 16S rRNA suggests that the two mutated nucleotides are in spatial proximity on opposite sides of a conserved stem structure in the 3' region of the molecule. Phylogenetic evidence is presented linking this conserved stem with spectinomycin resistance in chloroplasts. Perturbation of the stem is proposed to be the molecular-genetic basis for rRNA-dependent spectinomycin resistance.
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.
Microfluorometric analysis of the nuclear DNA contents of the somatic tissues of Arabidopsis thaliana has revealed extensive endoreduplication, resulting in tissues that comprise mixtures of polyploid cells. Endoreduplication was found in all tissues except those of the inflorescences and was developmentally regulated according to the age of the tissues and their position within the plant.
A protocol is described for the simple, rapid and efficient production of transgenic Arabidopsis plants. The procedure was developed using growth regulator regimes that promote adventitious embryogenesis during or immediately following Agrobacterium mediated transformation. Both the RLD and Columbia genotypes of Arabidopsis were transformed using slightly different growth regulator regimes. For the Columbia genotype two modifications of the protocol were identified which substantially improved regeneration. Cold treatment of the plants used as a source of root explants resulted in a three-fold increase in the number of morphogenic sectors produced. A more important modification was the inclusion of 25 mg/l silver nitrate (an inhibitor of ethylene action) to the medium used for shoot regeneration. This provided a ten-fold increase in the number of shoots produced. These procedures made it possible to obtain over 100 putative transformants of RLD or Columbia from a single 10 cm petri dish, within 2 or 4 weeks after exposure of root explants to the bacteria. When these were transferred to rooting media containing antibiotics, approximately 20% were able to root after kanamycin selection and 80% after hygromycin selection. All the rooted plantlets tested were shown to contain integrated donor DNA as determined by Southern blot analysis.
Excised roots of wild-type and nitrate-reductase deficient mutant Arabidopsis thaliana (L.) HEYNH. can be propagated as sustained root cultures in liquid medium. Culture initiation from a single seedling required a two-day indoleacetic acid treatment at 0.05 mg/l concentration. Indoleacetic acid facilitated subculture but was not essential for sustained growth. This procedure has allowed the clonal propagation of roots derived from individual wildtype and mutant seedlings for more than 21 months. The cultured roots retained their shoot regeneration ability; however, a controlled desiccation treatment was required to restore it to the level of freshly excised roots. The chromosome number remained diploid and no evidence for the accumulation of recessive mutations was observed. The cultured roots are competent for Agrobacterium-mediated transformation. The sustained root culture technology allowed the maintenance of transgenic tissues in which expression of a dominant, seed-lethal gene (seed-specific pea vicilin promoter fused to diphtheria toxin A chain gene) precluded generative propagation.
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.
To determine whether targeted DNA insertion into the tobacco plastid genome can be obtained without physical linkage to a selectable marker gene, we carried out biolistic transformation of chloroplasts in tobacco leaf segments with a 1:1 mix of two independently targeted antibiotic resistance genes. Plastid transformants were selected by spectinomycin resistance due to expression of an integrated aadA gene. Integration of the unselected kanamycin resistance (kan) gene into the same plastid genome was established by Southern probing in 20% of the spectinomycin-selected clones. Efficient cotransformation will facilitate targeted plastid genome modification without physical linkage to a marker gene.
The bacterial gene aad A encodes the enzyme aminoglycoside-3-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aad A gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.
The plastid genome of higher plants is relatively small, 120–230-kb in size, and present in up to 10,000 copies per cell. Standard protocols for the introduction of transforming DNA employ biolistic DNA delivery or polyethylene glycol treatment. Genetically stable, transgenic plants are obtained by modification of the plastid genome by homologous recombination, followed by selection for the transformed genome copy by the expression of marker genes that protect the cells from selective agents. Commonly used selective agents are antibiotics, including spectinomycin, streptomycin, kanamycin and chloramphenicol. Selection for resistance to amino acid analogues has also been successful. The types of plastid genome manipulations include gene deletion, gene insertion, and gene replacement, facilitated by specially designed transformation vectors. Methods are also available for post-transformation removal of marker genes. The model species for plastid genetic manipulation is Nicotiana tabacum, in which most protocols have been tested. Plastid transformation is also available in several solanaceous crops (tomato, potato, eggplant) and ornamental species (petunia, Nicotiana sylvestris). Significant progress has been made with Brasssicaceae including cabbage, oilseed rape and Arabidopsis. Recent additions to the crops in which plastid transformation is reproducibly obtained are lettuce, soybean and sugar beet. The monocots are a taxonomic group recalcitrant to plastid transformation; initial inroads have been made only in rice.
Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 micrograms/ml), SPC23 seedlings are resistant to low levels (50 micrograms/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.
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.
A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.
The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E. coli and in Agrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.
The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome ptDNA). The vectors
are based on the pUC119 plasmid which replicates in E.coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2- ptDNA sequences. Biolistic delivery of vector DNA,
followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, ∼ 1 transplastomic line per
bombarded sample. The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by homologous recombination events
via the flanking ptDNA sequences. The transplastomes thus generated are stable, and are maternally transmitted to the seed
progeny. The pPRV vector series targets insertions between the divergently transcribed trnV gene and rps12/7 operon. The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for
the study of transgene promoter activity.
We have developed efficient transformation protocols to modify each of the 500-10,000 plastid genome copies in a tobacco cell. The transforming DNA is introduced on the surface of microscopic tungsten particles by the biolistic process. Selection for transplastomes is by spectinomycin resistance based on expression of aminoglycoside-3"-adenyltransferase from a chimeric aadA gene in the transforming DNA. Manipulations that are now feasible include replacement of endogenous plastid genes with DNA sequences modified in vitro, targeted gene disruption, and insertion of reporter genes into the plastid genome. Alternative methods for plastid genome manipulations may be developed utilizing an extrachromosomal element which was identified during the transformation studies. Introduction of foreign genes under control of plastid gene expression elements results in duplication of endogenous regulatory sequences. A sensitive somatic assay to detect deletions via such direct repeats confirmed that these sequence duplications do not result in significant genome instability. The ability to transform plastids will facilitate the study of plastid gene regulation, and the application of genetic engineering to crop improvement.
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.
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.
Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an AatII site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia, and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 x 10(-5)) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.
A new vector, pFaadAII, for transformation of plastids of Nicotiana tabacum L. has been developed. It harbours a chimeric gene consisting of the aadA coding region from Escherichia coli, the 16S rDNA promoter from tobacco combined with a synthetic ribosome-binding site, a 500-bp fragment containing the 3' untranslated transcript region (UTR) of the Chlamydomonas rbcL gene and 3.75-kb (5') and 0.95-kb (3') tobacco plastome sequences allowing for targeting the foreign sequences to the intergenic region between the rpl32 and trnL genes of the tobacco plastome. The vector thus targets foreign sequences to the small single-copy region of the plastome, which has so far not been modified by transformation. Leaf protoplasts of Nicotiana tabacum L. were treated with polyethylene glycol (PEG) in the presence of the vector. The protocol for PEG treatment aiming at plastome transformation was optimized. Cell lines were cultured in the presence of spectinomycin and streptomycin using a novel and efficient protoplast culture and selection system. Regenerants were characterized by polymerase chain reaction (PCR) analysis, Southern hybridization and reciprocal crossing. The transformation procedure is described in detail and parameters influencing its efficiency are presented. Special effort is placed on analyzing suitable selection conditions. Only a proportion of the cell lines with a resistant phenotype could be confirmed by molecular analysis and/or reciprocal crossings to represent plastome transformants. Integration of the plastome specific aadA cassette into the nuclear genome accounted for a fraction of the resistant cell lines. Still, as many as 20-40 plastome transformants can be expected from the treatment of 10(6) protoplasts. Therefore, the improved protocol for PEG-mediated plastome transformation in combination with the new aadA-vector supplies a simple, reproducible and cost-efficient alternative to the biolistic procedure.
The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues. Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them. We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin. The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua. Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants. Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.
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.
Relative quantities of DNA in individual nuclei of stem and leaf epidermal cells of Arabidopsis were measured microspectrofluorometrically using epidermal peels. The relative ploidy level in each nucleus was assessed by comparison to root tip mitotic nuclei. A clear pattern of regular endopolyploidy is evident in epidermal cells. Guard cell nuclei contain levels of DNA comparable to dividing root cells, the 2C level (i.e., one unreplicated copy of the nuclear DNA). Leaf trichome nuclei had elevated ploidy levels of 4C, 8C, 16C, 32C, and 64C, and their cytology suggested that the polyploidy represents a form of polyteny. The nuclei of epidermal pavement cells were 2C, 4C, and 8C in stem epidermis, and 2C, 4C, 8C, and 16C in leaf epidermis. Morphometry of epidermal pavement cells revealed a direct proportionality between nuclear DNA level and cell size. A consideration of the development process suggests that the cells of highest ploidy level are developmentally oldest; consequently, the developmental pattern of epidermal tissues can be read from the ploidy pattern of the cells. This observation is relevant to theories of stomate spacing and offers opportunities for genetic analysis of the endopolyploidy/polyteny phenomenon.
Culture conditions were developed that induce Arabidopsis thaliana (L.) Heynh. root cuttings to regenerate shoots rapidly and at 100% efficiency. The shoots produce viable seeds in vitro or after rooting in soil. A transformation procedure for Arabidopsis root explants based on kanamycin selection was established. By using this regeneration procedure and an Agrobacterium tumor-inducing Ti plasmid carrying a chimeric neomycin phosphotransferase II gene (neo), transformed seed-producing plants were obtained with an efficiency between 20% and 80% within 3 months after gene transfer. F(1) seedlings of these transformants showed Mendelian segregation of the kanamycin-resistance trait. The transformation method could be applied to three different Arabidopsis ecotypes. In addition to the neo gene, a chimeric bar gene conferring resistance to the herbicide Basta was introduced into Arabidopsis. The expression of the bar gene was shown by enzymatic assay.
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