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Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system
Abstract
Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.
... Crossover events in the two flanks replace the IR with the selectable marker gene for plastid transformation, a chimeric aadA gene conferring spectinomycin resistance 23 (Fig. 1b). The aadA cassette was equipped with loxP sites immediately upstream and downstream to enable post-transformation marker removal by site-specific recombination mediated by the Cre recombinase 24 (Fig. 2b,c). As a control for neutrality of the marker insertion site in the chloroplast genome, an additional vector was constructed (aadA control) that leaves both IRs intact and solely integrates the aadA cassette (with its flanking loxP sites) into the border between the IR and the SSC (Fig. 1). ...
... The removal of the selectable marker gene via Cre-mediated loxP site-specific recombination was conducted as previously described 24,51 . Briefly, marker gene excision was achieved by pollination with a homozygous Cre-expressing line that contains a gentamycin resistance marker 24 . ...
... The removal of the selectable marker gene via Cre-mediated loxP site-specific recombination was conducted as previously described 24,51 . Briefly, marker gene excision was achieved by pollination with a homozygous Cre-expressing line that contains a gentamycin resistance marker 24 . The progeny resulting from this cross were selfed, and the next generation was tested for the absence of the aadA marker gene and the recombinase gene (linked to the gentamycin resistance gene). ...
The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.
... The CRE-loxP recombination system is used in the other method to eliminate marker genes from transgenic plants. A 34-bp sequence from the P1 bacteriophage is LoxP [86], that is identified by a 38.5-kDa CRE protein expressed from the cre gene [87]. The loxP component is made up of two 13-bp repetitions separated by an 8-bp spacer region. ...
... Recombination reactions are regulated by CRE recombinase. Such system was employed in plants to eliminate genes from the nucleus [88] and also from plastids [87] by either constant or temporary expression of the cre gene by the nucleus. Nevertheless, such strategy was troubling because of the existence of pseudo-loxP locations in the genome of plastid, that caused the depletion of ptDNA owing to increased recombination activity [87]. ...
... Such system was employed in plants to eliminate genes from the nucleus [88] and also from plastids [87] by either constant or temporary expression of the cre gene by the nucleus. Nevertheless, such strategy was troubling because of the existence of pseudo-loxP locations in the genome of plastid, that caused the depletion of ptDNA owing to increased recombination activity [87]. A further alternative strategy of the CRE/loxP method is the use of phiC31 phage site-specific integrase (Int), that facilitates recombination between bacterial (attB) and phage (attP) attachment sites. ...
th 13 Photosynthesis seems to be a central process which converts the sunlight to energy, thereby promotes plant proliferation and development, and leads to the maintenance of life on the earth by using water to introduce oxygen into the environment. Increased plant photosynthesis has recently been revitalized as a strategy to increase yields of crops and solve global food problems. Photosynthesis, thus, has become a primary focus of genetic engineering. Multiple attempts are ongoing to improve photosynthesis in order to overcome the obstacles of developing food and fuel demand in speedily evolving global environments. In order to improve photosynthetic capacity, several transgenes have been inserted into the plastid or nuclear genomes. Plant transformation has become a key experimental approach in plant science as well as a functional approach for transgenic plant growth. There are several confirmed strategies for the effective integration of desired genes into the genomes of various plants. This review article explains the importance of the genetic engineering in the modification and improvement of photosynthesis and its potential utilization in increasing crop yields.
... The CRE-loxP recombination system is used in the other method to eliminate marker genes from transgenic plants. A 34-bp sequence from the P1 bacteriophage is LoxP [86], that is identified by a 38.5-kDa CRE protein expressed from the cre gene [87]. The loxP component is made up of two 13-bp repetitions separated by an 8-bp spacer region. ...
... Recombination reactions are regulated by CRE recombinase. Such system was employed in plants to eliminate genes from the nucleus [88] and also from plastids [87] by either constant or temporary expression of the cre gene by the nucleus. Nevertheless, such strategy was troubling because of the existence of pseudo-loxP locations in the genome of plastid, that caused the depletion of ptDNA owing to increased recombination activity [87]. ...
... Such system was employed in plants to eliminate genes from the nucleus [88] and also from plastids [87] by either constant or temporary expression of the cre gene by the nucleus. Nevertheless, such strategy was troubling because of the existence of pseudo-loxP locations in the genome of plastid, that caused the depletion of ptDNA owing to increased recombination activity [87]. A further alternative strategy of the CRE/loxP method is the use of phiC31 phage site-specific integrase (Int), that facilitates recombination between bacterial (attB) and phage (attP) attachment sites. ...
Photosynthesis seems to be a central process which converts the sunlight to
energy, thereby promotes plant proliferation and development, and leads to
the maintenance of life on the earth by using water to introduce oxygen into
the environment. Increased plant photosynthesis has recently been
revitalized as a strategy to increase yields of crops and solve global food
problems. Photosynthesis, thus, has become a primary focus of genetic
engineering. Multiple attempts are ongoing to improve photosynthesis in
order to overcome the obstacles of developing food and fuel demand in
speedily evolving global environments. In order to improve photosynthetic
capacity, several transgenes have been inserted into the plastid or nuclear
genomes. Plant transformation has become a key experimental approach in
plant science as well as a functional approach for transgenic plant growth.
There are several confirmed strategies for the effective integration of
desired genes into the genomes of various plants. This review article
explains the importance of the genetic engineering in the modification and
improvement of photosynthesis and its potential utilization in increasing crop
yields.
... For marker elimination, homoplasmic transplastomic plants were crossed to nuclear-transgenic plants expressing a plastid-targeted Cre recombinase (Corneille et al. 2001). The resulting seeds were surface sterilized and cultivated on MS medium containing 20 g/L sucrose and 500 mg/L spectinomycin. ...
... The griffithsin cassette was then integrated into a standard plastid transformation vector (Zhou et al. 2008) containing a chimeric spectinomycin resistance gene (aadA) as selectable marker. In this vector, the aadA gene is flanked by loxP sites to facilitate post-transformation removal of the antibiotic resistance marker (Fig. 1a; Corneille et al. 2001). ...
... Use of a loxP-flanked ('floxed') version of the aadA marker gene in our transformation construct (Fig. 1a) allowed excision of the aadA cassette by site-specific recombination triggered by a nucleus-encoded Cre recombinase that is post-translationally imported into plastids (Corneille et al. 2001). To this end, we crossed homoplasmic transplastomic Nt-grft lines (maternal parent) to a nuclear transgenic Cre-expressing line (pollen donor). ...
Key message:
The potent anti-HIV microbicide griffithsin was expressed to high levels in tobacco chloroplasts, enabling efficient purification from both fresh and dried biomass, thus providing storable material for inexpensive production and scale-up on demand. The global HIV epidemic continues to grow, with 1.8 million new infections occurring per year. In the absence of a cure and an AIDS vaccine, there is a pressing need to prevent new infections in order to curb the disease. Topical microbicides that block viral entry into human cells can potentially prevent HIV infection. The antiviral lectin griffithsin has been identified as a highly potent inhibitor of HIV entry into human cells. Here we have explored the possibility to use transplastomic plants as an inexpensive production platform for griffithsin. We show that griffithsin accumulates in stably transformed tobacco chloroplasts to up to 5% of the total soluble protein of the plant. Griffithsin can be easily purified from leaf material and shows similarly high virus neutralization activity as griffithsin protein recombinantly expressed in bacteria. We also show that dried tobacco provides a storable source material for griffithsin purification, thus enabling quick scale-up of production on demand.
... The third approach uses the CRE-loxP recombination system to excise marker genes from transgenic plants. The loxP is a 34-bp sequence from P1 bacteriophage [108], which is recognized by a 38.5-kDa CRE protein encoded by the cre gene [109]. The loxP element consists of two 13-bp repeats, separated by an 8-bp spacer region. ...
... The recombination reaction is mediated by CRE recombinase. This system has been used in plants to remove genes from the nucleus [110] as well as plastids [109] either by stable or transient expression of cre gene via nucleus. However, this approach was problematic due to the presence of pseudo loxP sites in the plastid genome, which resulted in the loss of ptDNA by enhanced recombination activity [109]. ...
... This system has been used in plants to remove genes from the nucleus [110] as well as plastids [109] either by stable or transient expression of cre gene via nucleus. However, this approach was problematic due to the presence of pseudo loxP sites in the plastid genome, which resulted in the loss of ptDNA by enhanced recombination activity [109]. ...
An alarming increase in the human population necessitates doubling the world food production in the next few decades. Although a number of possible biotechnological measures are under consideration, central to these efforts is the development of transgenic crops to produce more food, and the traits with which plants could better adapt to adverse environmental conditions in a changing climate. The emergence of new tools for the introduction of foreign genes into plants has increased both our knowledge and the capacity to develop transgenic plants. In addition, a better understanding of genetic modifications has allowed us to study the impact that genetically modified crop plants may have on the environment. This article discusses different techniques routinely used to carry out genetic modifications in plants while highlighting challenges with them, which future research must address to increase acceptance of GM crops for meeting food security challenges effectively.
... While the first published strategy to remove the selectable marker utilized homologous recombination (Iamtham and Day, 2000), the use of specific recombinases has become a viable alternative to produce marker-free transplastomic plants. Cre/lox was the first site-specific recombination system utilized to excise the SMG from the plastid genome (Corneille et al., 2001;Hajdukiewicz et al., 2001). Cre activity was introduced by nuclear transformation and marker-free transplastomic plants were obtained in tissue culture. ...
... Cre was then segregated away in the progeny. Unfortunately, Cre-mediated marker excision can also facilitate the undesirable deletion of ptDNA sequences via the recognition of repeated nonlox sequences resulting in mutations of target plant plastid genome (Corneille et al., 2001;Hajdukiewicz et al., 2001;Tungsuchat et al., 2006). The large serine subfamily recombinases phiC31 and Bxb1 were also shown to excise the SMGs (Kittiwongwattana et al., 2007;Shao et al., 2014). ...
... Evidence of complete excision was obtained in all F 1 progeny of each successful cross. These results are similar to the successful excision observed in Cre-lox (Corneille et al., 2001) and Bxb1 systems (Shao et al., 2014) and could be classified as a strong activator owing to the observed 100% excision in the seedling tested. We sequenced the 0.76-kb (ParA-MRS) and 0.89-kb (CinH-RS2) PCR amplicons from the F 1 DNA of six independent crosses and found perfectly conserved MRS or RS2 sites in each one (Figure 1d, e). ...
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 first group of methods exploit native properties of the plastid genetic system including the predominance of homologous DNA recombination [29] and sorting out of marker-free plastomes from a heteroplasmic mixture of plastomes. The second group of methods involves the introduction of foreign site-specific recombinases and their target sites into plastids [22][23][24][25][26]. Methods exploiting the natural DNA maintenance mechanisms in plastids include (1) marker excision using directly repeated DNA sequences (DNA direct repeats), (2) transient cointegration of a marker gene followed by its excision [27], (3) cotransformation and integration of marker and herbicide resistance genes into different plastomes followed by segregation of marker-free herbicide resistant plastomes from wild type and other transgenic plastomes [28]. ...
... This suggests that recombination between direct repeats is limited by Rec A activity in wild type Chlamydomonas chloroplasts. The frequency of recombination between short direct repeats of~120 bp appears to be stimulated by the presence of Cre site-specific recombinase in plastids [22,23]. Similarly, recombination between attP and attB sites in transgenic plastid DNA by phiC31 site-specific recombinase stimulated recombination between 84 bp direct repeats of the rrn promoter [39]. ...
Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.
... In this study, the aadA gene was used to select for integration events. For this reason aadA had been removed from R1 recipient tobacco lines using the Cre/loxP recombinase system [16][17][18]. To develop marker-free R1 recipient lines, the loxP sites were cloned on either side of the aadA gene. ...
... To develop marker-free R1 recipient lines, the loxP sites were cloned on either side of the aadA gene. The aadA gene was removed by providing nuclearencoded recombinase [17] and the resulting tobacco lines were named R2 recipient lines; the R2 lines contain only an attB site along with the reconstituted loxP site upon aadA removal. Moreover, two helper plasmids were used to provide INT: in one of them, the translation of int-derived mRNA was dependent on the creation of a translation initiation site (AUG) by mRNA editing, whereas no editing was required in the second plasmid. ...
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).
... An additional strategy is marker gene excision, which can be accomplished through direct repeat-mediated recombination (30,58) or the use of the CRE-lox system (17). More recently, the site-specific Bxb1 recombinase has been used with attP/attB sites to precisely excise DNA from the plastid genome in the widely used trnI/trnA region, making it a valuable tool for future studies (116). ...
... The most desirable selectable markers would utilize the plant's own genes and metabolic pathways (30). Alternatively, the plastid's highly active homologous recombination machinery (30) or the use of exogenous recombinases could be exploited to excise a selectable marker after achieving homoplasmy (17,116). In particular, using Bxb1 recombinase and attP/attB sites to precisely remove DNA from within the trnI/trnA intergenic spacer shows great promise for this widely used site (116). ...
Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer's, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy. Codon optimization utilizing valuable information from sequenced chloroplast genomes enhanced expression of eukaryotic human or viral genes in chloroplasts and offered unique insights into translation in chloroplasts. Support from major biopharmaceutical companies, development of hydroponic production systems, and evaluation by regulatory agencies, including the CDC, FDA, and USDA, augur well for advancing this novel concept to the clinic and revolutionizing affordable healthcare.
... The pL3-PrrnG10LTrxf vector (Fig. 1a) was used for the plastid transformation (Sanz- Barrio et al. 2013). This vector includes the trx f coding sequence under the control of the rrn promoter fused to the leader sequence of the phage T7 gene 10 (G10L), containing also the selectable spectinomycin resistance gene (aadA) flanked by two lox sequences suitable for Cre-mediated gene marker excision (Corneille et al. 2001). The plastid transformation vector targets the insertion of transgenes in the duplicated inverted repeat region, between the 16S3 0 /trnV and 3 0 rps12 genes. ...
... Since the Cre-lox site-specific recombination system has already been efficiently used to obtain marker-free transplastomic plants (Corneille et al. 2001), transplastomic T1 plants were nuclearly transformed with the cre gene from the P1 phage via A. tumefaciens. The transit peptide of the tobacco RuBisCO small subunit (SSU) fused to the N-terminus of Cre recombinase (SSU-Cre) targeted Cre to the plastid ( Supplementary Fig. S1a), allowing the excision of the lox-flanked aadA gene from the plastid genome. ...
... (1) A CRE-loxP system has been applied to excise markers on stable transformants (Corneille et al. 2001), which is based on the addition of recombinase-recognition sites ( loxP) flanking the selectable marker. The expression of a Cre gene is subsequently induced by a transient expression approach (Lutz et al. 2006), inducing a loxP-mediated excision of the marker gene in a site-specific recombination event. ...
... (3) The use of co-integrated vectors has been proposed by Klaus et al. (2004), where one vector having a single homologous recombination and a marker gene located outside of the recombination region allows for the selection of transformants, but in a subsequent step generates an unstable arrangement due to the presence of direct repeats, leading to gene marker excision. Therefore, molecular tools for developing transplastomic plants devoid of selection markers constitute an attractive alternative for the field of plant-based vaccine development (Corneille et al. 2001). ...
... Countries like India have approved only non-edible plants like Bt cotton for commercial cultivation. Although tools were introduced to eliminate the marker gene from transgenic plants (Gleave et al., 1999;Matthews et al., 2001;Corneille et al., 2001), the presence of the foreign gene in the host chromosome of transgenic plants caused concern for commercial cultivation. ...
Genome editing tools based on CRISPR/Cas system have been posed to solve many issues in agriculture and improve food production. Genetic engineering by Agrobacterium-mediated transformation has helped to impart specific traits straightaway in many crops. Many GM crops have also reached the field for commercial cultivation. Genetic engineering requires mostly a transformation protocol often mediated by Agrobacterium to insert a specific gene at a random locus. Genome editing with CRISPR/Cas system is a more precise technique for the targeted modification of genes/bases in the host plant genome. Unlike the conventional transformation system, wherein elimination of marker/foreign gene was possible only post-transformation, CRISPR/Cas system could generate transgene-free plants by delivering CRISPR/Cas reagents such as the Cas protein and gRNA(s) as preassembled to form ribonucleoproteins (RNPs) into plant cells. CRISPR reagent delivery might be helpful to overcome issues with plants that are recalcitrant to Agrobacterium transformation and the legal hurdles due to the presence of the foreign gene. More recently, the grafting of wild-type shoots to transgenic donor rootstocks developed by the CRISPR/Cas system has reported transgene-free genome editing. CRISPR/Cas system also requires only a small piece of guide RNA (gRNA) besides Cas9 or other effectors to target a specific region in the genome. So this system has been projected to be a key contributor to future crop breeding. In this article, we recap the main events of plant transformation, compare the difference between genetic transformation and CRISPR-mediated genome editing, and draw insights into the future application of the CRISPR/Cas system.
... This article is protected by copyright. All rights reserved have been generated through the use of recombinases (Corneille et al., 2001;Hajdukiewicz et al., 2001), loop-out recombination (Ruf et al., 2007), transient cointegration of two chloroplast vectors (Klaus et al., 2004), or direct repeat recombination of transgenes integrated into the large single copy region of tobacco (Iamtham and Day, 2000), soybean (Dufourmantel et al., 2007) or the inverted repeat region of lettuce Daniell et al., 2019b;Kumari et al., 2019;Park et al., 2020;Singh et al., 2021). As demonstrated in this work, it is possible to design integrating episomal constructs, where the desired transgenes are incorporated into the native plastome through HR, while the selection genes are expressed on the episomal construct. ...
In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over thirty‐years‐old. Here we show the design‐build‐test of a novel synthetic genome structure that does not disturb the native plastome: the “mini‐synplastome.” The mini‐synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini‐synplastome in plants was developed in vitro to meet the following criteria: 1) episomal replication in plastids; 2) facile cloning; 3) predictable transgene expression in plastids; 4) non‐integration of vector sequences into the endogenous plastome and; 5) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini‐synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker‐free plastid engineering, and provide an unparalleled platform for one‐step metabolic engineering in plants.
... Nonetheless, the results obtained by Yoo et al. [65] in Chlamydomonas chloroplasts open the door to engineering organelle genomes. They demonstrated, for the first time, the editing of the chloroplast genome with Cas9 to integrate DNA with high precision in the plastome; although these results are highly promising, plastid editing is still underexplored [63], and the selection of marker genes must still be eliminated using the Cre/loxP system [66,67], phiC31 phage site-specific integrase [68], and the directly repeated DNA sequences [62,69]. ...
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, the selectable marker gene especially the old markers remain a public concern (Schaart et al. 2004). Using CRE-lox recombinase CoDA selectable marker flanked with two directly oriented lox sites, the highly efficient elimination of the marker gene (Table 3) introduced through pollination was reported (Corneille et al. 2001). In another research report using the CRISPR/Cas 9 system, depigmentation and leaf size expansion have been done by a targeted mutation on NtPDS and NsSTF1 genes, respectively. ...
The search for technologies for crop improvement has been a continuous practice to address the food insecurity to the growing human population with an ever-decreasing arable land and dynamic climate change around the world. Considering the potential technologies for crop improvement could close the rooms of poverty in developing countries in particular and around the globe at large. This review aimed to assess the site-directed mutation creation methods and show the potential tools for future crop improvement programs. Site-directed mutagenesis was found to be an efficient process to create targeted mutation on cereal crops, horticultural crops, oilseed crops, and others. Agronomic traits such as yield, quality, and stress tolerance have been improved using site-directed mutagenesis. Besides, selectable marker elimination was also reported from transgenic crops by targeted mutation. Most of the reports on site-directed mutagenesis is focusing on cereal crops (58.339%) followed by horticultural crops (22.92%). Among the four mutagenic tools that have been reported, the CRISPR/Ca9 technology was found to be frequently used (66.67%) followed by TALENs. This tool is potential, since it is efficient in creating targeted mutagenesis and less likely off-target effect, so it is repeatedly used in different research works. TALENs were used usually to knockout genes with bad traits. Moreover, the mutation created by mutagenic tools was found to be efficient, and the mutated traits proved to be heritable to generations. Hence, site-directed mutagenesis by the CRISPR/Cas9 system is advisable for agricultural development, thereby ensuring food sustainability around the world.
... However, the selectable marker gene especially the 319 old markers remain a public concern (80). Using CRE-lox recombinase CoDA selectable 320 marker flanked with two directly oriented lox sites, the highly efficient elimination of the 321 marker gene (Table 3) introduced through pollination was reported (81). By another research 322 report, depigmentation and leaf size expansion has been done by a targeted mutation on 323 NtPDS and NsSTF1 genes respectively by using the CRISPR/Cas 9 system. ...
The search for technologies for crop improvement has been a continuous practice to address the food insecurity to the growing human population with an ever decreasing arable land and dynamic climate change around the world. Considering the potential technologies for crop improvement could close the rooms of poverty in developing countries in particular and around the globe at large. This review aimed to assess the site-directed mutation creation methods and to show the potential tools for future crop improvement programs. Site-directed mutagenesis was found to be an efficient process to create targeted mutation on cereal crops, horticultural crops, oilseed crops, and others. Agronomic traits such as yield, quality, and stress tolerance have been improved using site-directed mutagenesis. Besides, selectable marker elimination was also reported from transgenic crops by targeted mutation. Most of the reports on site-directed mutagenesis is focusing on cereal crops (58.339%) followed by horticultural crops (22.92%). Among the four mutagenic tools that have been reported, the CRISPR/Ca9 technology was found to be frequently used (66.67%) followed by TALENs. This tool is potential since it is efficient in creating targeted mutagenesis and less likely off-target effect, so it is repeatedly used in different research works. TALENs were used usually to knockout genes with bad traits. Moreover, the mutation created by mutagenic tools found to be efficient, and the mutated traits proved as it was heritable to generations. Hence, site-directed mutagenesis by the CRISPR/Cas9 system is advisable for agricultural development thereby ensuring food sustainability around the world.
... Homologous recombination is efficient in chloroplasts (Corneille et al. 2001) 13.14. The marker-free GMP can be then selected by progeny segregation. ...
Somaclonal variation, ploidy stability, DNA fingerprinting and characterization of germplasm, modes of plant gene modification, and Agrobacterium-mediated and biolistic transformation in mono and dicotyledonous plants with several examples of actual lab-based detailed techniques are described in this chapter. Although the gene transfer mechanisms remain largely unknown, great progress has been made in the practical implementation of transformation protocols for both dicotyledonous and monocotyledonous plants. Particularly important is the extension of this single-cell transformation methodology to monocotyledonous plants. This advance has biological and practical implications. Transgenic carrot as potential source of edible vaccines developed in our lab is given in details. Furthermore, the use of reporter genes for detection of transformation efficiency, protein localization study, and tagging of beneficial microbes for determination of infestation the plant root are demonstrated. Gene silencing by means of RNA interference (RNAi) technology offers the possibility to inhibit expression of entire gene families. This aim will be achieved by selecting nucleotide regions of high similarity often found within coding regions. The creation of a double-strand break at a specific location within a genome by using ZFN, TALEN, and CRISPR-CAS system has been described. For better understanding of gene technology, a few examples of our own research program will be described.
... Alternatively, selectable markers may be selectively removed after the construction of the transgenic plant (Iamtham and Day, 2000). For example, the marker can be flanked by loxP sequences and eliminated from the genome using Cre-mediated recombination (Corneille et al., 2001). ...
... Further advantages lie in a high level of accumulation of the protein encoded by the transgene, the direct use of prokaryotic sequences, which can be organized in an operon, and site-specific integration through homology-directed repair, all of which are properties related to the prokaryotic origin of the organelle genome (Maliga 2004). Furthermore, excision of the selectable marker gene using either the Cre/lox system of natural intra-chromosomal recombination has been shown to be possible (Corneille et al. 2001). Although the reduced size of the proplastids in embryogenic cells competent for regeneration appear to preclude their use for plastid transformation in rice, the generation of homoplastic rice plants has been reported (Lee et al. 2006). ...
Rice, the staple food for more than half of humankind and the model monocot crop, was the first cereal in which efficient gene transfer procedures were implemented: 30 years ago, the first transgenic rice plant was regenerated following direct gene transfer to cell suspension‐derived protoplasts. Shortly thereafter, transgenic plants were regenerated from zygotic embryo‐derived cells following their subjection to micro‐projectile acceleration and Agrobacterium‐mediated transfection treatments. The high efficiency of transfer deoxyribonucleic acid (T‐DNA) integration in seed embryo‐derived cells of rice has allowed the transfer of genes of agronomical relevance and the generation of large collections of insertion lines and has provided a key contribution to deciphering the function of more than 2000 rice genes. The high efficiency of T‐DNA integration in seed embryo‐derived cells of rice also permitted the first implementation of gene targeting and knock‐in (KI) events, relying on the albeit very low natural frequency of homology‐directed repair (HDR) in the rice genome. In the late 2000s, the advent of site‐directed nucleases (SDNs) that induce either single or double‐strand breaks at a high frequency and their rapid application to rice permitted routine targeted mutagenesis, which can be multiplexed to simultaneously alter several targets or create deletions, and base and gene editing (e.g. correction of amino acids). Currently, the challenge remains to attain a high frequency of KI and replacement of long stretches of DNA for protein domain or coding sequence swapping. We present herein a historical perspective of the advances that have been readily implemented to determine the function of rice genes and to manipulate traits of agronomical relevance. Two main bottlenecks remain to be alleviated in rice genomic engineering: the low frequency of HDR and the genotype dependence of gene transfer efficiency. Alleviation of these bottlenecks is needed to reach the potential of intra‐ and interspecific gene replacement and SDN‐mediated multiplex editing of alleles in elite materials, which will assist in the breeding and deployment of rice cultivars embedded in sustainable and climate‐smart agricultural practices.
... En transformación plastidial además, existen varias alternativas para eliminar los genes marcadores de selección (Ye et al., 2003) para obtener plantas. Entre los métodos disponibles se encuentran: (1) el uso de recombinasas como CRE (Corneille et al., 2001) o Int (Kittiwongwattana et al., 2007), (2) la co-integración transitoria del gen marcador (Klaus et al., 2004), (3) la recombinación entre secuencias repetidas en la misma orientación (Fischer et al., 1996;Iamthan y Day, 2000;Kode et al., 2006;Dufourmantel et al., 2007) o (4) la segregación por cotransformación (Ye et al., 2003). ...
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.
... This approach is now well-established for engineering of plant chloroplasts, but the method has been demonstrated only for marker excision from the C. reinhardtii nucleus [99] and has yet to be demonstrated for the chloroplast. Various recombinases have been shown to work in the tobacco chloroplast, including Cre recombinase [100], phiC31 [101] and Bxb1 [102]. These systems all work similarly and employ a two-step approach: a selectable marker flanked by recognition sequences specific to the recombinase is used for chloroplast transformation, and then, after selection of transplastomic lines, a gene encoding a chloroplast-targeted version of the recombinase is introduced into the plant nucleus. ...
Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.
... (CrPpsbA), and followed by the 3 ′ UTR from the C. reinhardtii rbcL gene (CrTrbcL). The cassette (1,647 bp) is flanked by two loxP sites to facilitate marker gene excision by Cre-mediated site-specific recombination (Corneille et al., 2001;Zhou et al., 2008). The reporter gene gfp is controlled by the tobacco plastid rRNA operon promoter combined with the 5 ′ UTR from gene10 of bacteriophage T7 (NtPrrn:T7g10) (Kuroda and Maliga, 2001), and the 3 ′ UTR from the E. coli ribosomal RNA operon rrnB (TrrnB). ...
Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5′ untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3′UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.
... The engineered chromosomes should be amenable for in vivo manipulations. In plants, site-specific recombinases have been used to generate gene deletions, insertions, inversions and the resolution of tandemly arrayed transgenes (Dale and Ow, 1990;Lloyd and Davis, 1994;Qin et al., 1994;Sauer, 1994;Albert et al., 1995;Corneille et al., 2001). The engineered chromosome can provide a defined recipient locus onto which subsequent genes of interest are introduced, enabling genetic manipulation via site-specific recombination systems. ...
Engineered minichromosomes could be stably inherited and serves as a platform for simultaneously transferring and stably expressing multiple genes. Chromosomal truncation mediated by repeats of telomeric sequence is a promising approach for the generation of minichromosomes. In the present work, direct repetitive sequences of Arabidopsis telomere were used to study telomere-mediated truncation of chromosomes in Brassica napus. Transgenes containing alien Arabidopsis telomere were successfully obtained, and Southern blotting and fluorescence in situ hybridization (FISH) results show that the transgenes successfully resulted in chromosomal truncation in B. napus. In addition, truncated chromosomes were inherited at rates lower than that predicted by Mendelian rules. To determine the potential manipulations and applications of the engineered chromosomes, such as the stacking of multiple transgenes, the Cre/lox and FRT/FLP recombination systems, both amenable for genetic manipulations through site-specific recombination in somatic cells, were tested for their ability to undergo recombination in B. napus. These results demonstrate that alien Arabidopsis telomere are able to mediate chromosomal truncation in B. napus. This technology would be feasible for chromosomal engineering and for studies on chromosome structure and function in B. napus. This article is protected by copyright. All rights reserved.
... Additionally, the public fears that plants containing antibiotic resistance genes may inactivate oral doses of the antibiotic (Daniell, Wiebe, and Fernandez-SanMillan, 2001). Various methods such as co-transformation (Carrer and Maliga, 1995), loop-out recombination (Lamtham and Day, 2000), CRE/lox system (Corneille et al., 2001;Hajdukiewicz, Gilbertson, and Staub, 2001), BADH (Daniell, Wiebe, and Fernandez-SanMillan, 2001) and the use of transiently cointegrated selection genes (Klaus et al., 2004) have all offered solutions for these concerns. Selection of homoplasmic transformants is random in loop-out recombination and the introduction and removal of the CRE recombinase require additional transformation and sexual crossing (Klaus et al., 2004). ...
Chloroplast genetic engineering offers a number of unique advantages, including a high-level of 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, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. 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. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.
... As with direct repeat-mediated loop-out recombination, the approach requires a secondary marker, i.e., the resulting lines are not "markerfree", and the expression of the secondary marker might constitute an unnecessary metabolic burden. Hajdukiewicz et al. (2001) and Corneille et al. (2001) independently and simultaneously introduced CRE recombinase-mediated marker removal from transplastomic tobacco. The expression of the CRE protein-encoding gene, derived from the P1 bacteriophage, leads to insertion or excision of sequence elements, provided that recognition elements, loxP sites, are present on the recombination substrate molecules. ...
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.
... Marker genes are helpful for transformation, but on the other hand, there are some risks associated with their use, i.e. spontaneous mutations or threat of gene escape. Efforts to produce the marker free transgenic and by using the reduced risk strategies, these risks can be overcome to some extent i.e., CRE-loxP site specific recombination system, involving AC-Ds elements are under study, which helps to eliminate the marker gene once the transformation is successfully completed [18]. Transient co-integration, site-specific recombinases and their target sites can also be employed effectively for producing marker free plants [19]. ...
Engineering microalgae has opened a new era for plant biologists and biotechnologists. Microalgae had been proved as a promising candidate for the production of biopharmaceuticals, nutraceuticals, antioxidants, antimicrobial and antiviral compounds, in dyeing and food industry as well for biofuel production. Genetic transformation of some important microalgae has been successful, but several other potential microalgae species still need scientific attention. The success of the genetic transformation depends mainly on the utilization of the selectable and screenable markers. Like for other higher crop plants, several useful markers have been reported for microalgae transformation. In this follow-up, we compared different marker genes for genetic engineering of approximately all the industrially important microalgae. We have discussed the expression host, the targeted genome, appropriate selection agent, as well as the transformation method. Genetic transformation is an expensive and labor intensive process and this review will aid to shorten the time span by providing a database of appropriate markers for microalgae research which could serve as a guide for those involved in the genetic engineering of microalgae.
... The marker gene is then removed by the introduction of a gene encoding a plastid-targeted recombinase in the plant nucleus, which eventually enters all plastids and simultaneously excises the marker genes flanked by directly oriented target sites [49]. Two of such recombinases, the Cre recombinase, derives from the P1 bacteriophage, and which excises target sequences flanked by directly oriented 34 bp loxP sites [52,53,54,55,48] and the ΦC31 phage integrase, Int [56,57] have been used successfully to achieve marker-free transplastomic plants. ...
Plastid transformation offers a viable alternative to nuclear transformation because of its numerous advantages. It was against this backdrop that various groups of researchers have been exploiting this group of sub-cellular organelles, over the last two decades, for the genetic engineering of agronomic traits and metabolic pathways, as well as for molecular farming for the production of plant-derived high-valued biopharmaceuticals and industrial proteins. In this short review, we discuss the evolution and development of this technology with respect to the current state-of-the-art, which places it in high pedestal as a cost effective and safe production technology for high quality molecular farming products as well as a highly efficient method to create new metabolic pathways and improve the agronomic traits.
... So far, cotransformation with antibiotic and herbicide resistance genes allows for the recovery of marker-free transformed plants (Dufourmantel et al., 2007). A more powerful approach relies on transient expression of phage recombinases (CRE-lox and phage integrase) to remove the selection marker (via flanking target sequences) once the plant reaches homoplasticity (Corneille et al., 2001;Lutz et al., 2004Lutz et al., , 2006. The specific challenges associated with protoplast preparation and achieving homoplasticity are beyond the scope of this article, but have been reviewed extensively elsewhere (Lutz and Maliga, 2007;Purton, 2007;Day and Goldschmidt-Clermont, 2011;Maliga and Bock, 2011;Bock, 2014). ...
The chloroplasts found in plants and algae, and photosynthetic microorganisms such as cyanobacteria, are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals, as well as complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the production levels to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the synthetic biology tools available and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts. This article is protected by copyright. All rights reserved.
... Therefore, elimination of selectable marker genes from transgenic plants is highly recommended. Different methods including marker excision (Cre-lox site-specific recombination system) and cotransformation followed by independent segregation of antibiotic and herbicide genes have been successfully used to produce marker free transgenic plants (Ye et al., 2003;Corneille et al., 2001;Hajdukiewicz et al., 2001;Zou, 2001). ...
Presence of antibiotic resistance markers has always been considered as one of the main safety concerns in transgenic plants and their derived products. Elimination of antibiotic selectable markers from transgenics is a major hurdle for finding efficient and safe candidates. Herbicide tolerance genes might be attractive alternatives. In this study, a variant form of the 5-enoylpyruvyl shikimate-3-phosphate synthase (EPSPS) gene that harbors glycine at position 96 to alanine and alanine 183 to threonine substitutions and confers higher resistance to the broad-spectrum herbicide, glyphosate, was substituted against the spectinomycin resistant gene as a sole selectable marker for plastid transformation of Nicotiana tabacum. Plastid transformation was carried out using the biolistic delivery procedure while delivery parameters such as rupture disk pressure, bombardment distance, etc had been optimized first. A previous study showed that the glyphosate herbicide imposes lethal effects on the structure and integrity of the plastid membrane, even at low concentrations. In order to overcome this problem, a modified procedure for selection of transplastomic cells was used. A long preculture incubation period followed by a gradual increased in glyphosate concentration led to sufficient expression of the transgene. Tolerant calli were thus regenerated through direct selection of transformed plastids in the presence of the glyphosate.
... This is an exceptional advantage because it allows the production of transgenic plants without the insertion of antibiotic resistance genes, eliminate any possibility of antibiotic resistance gene flow to neighboring crop fields or to crop wild relatives growing near the transgenic crops. Moreover, it permits the recycling of selectable marker genes, which can be reused in a new genetic transformation event in the same transgenic plant (Carrer et al., 1993;Svab and Maliga, 1993;Corneille et al., 2001;Barone et al., 2009;Li et al., 2011). ...
During the evolution of the eukaryotic cell, plastids, and mitochondria arose from an endosymbiotic process, which determined the presence of three genetic compartments into the incipient plant cell. After that, these three genetic materials from host and symbiont suffered several rearrangements, bringing on a complex interaction between nuclear and organellar gene products. Nowadays, plastids harbor a small genome with ∼130 genes in a 100–220 kb sequence in higher plants. Plastid genes are mostly highly conserved between plant species, being useful for phylogenetic analysis in higher taxa. However, intergenic spacers have a relatively higher mutation rate and are important markers to phylogeographical and plant population genetics analyses. The predominant uniparental inheritance of plastids is like a highly desirable feature for phylogeny studies. Moreover, the gene content and genome rearrangements are efficient tools to capture and understand evolutionary events between different plant species. Currently, genetic engineering of the plastid genome (plastome) offers a number of attractive advantages as high-level of foreign protein expression, marker gene excision, gene expression in operon and transgene containment because of maternal inheritance of plastid genome in most crops. Therefore, plastid genome can be used for adding new characteristics related to synthesis of metabolic compounds, biopharmaceutical, and tolerance to biotic and abiotic stresses. Here, we describe the importance and applications of plastid genome as tools for genetic and evolutionary studies, and plastid transformation focusing on increasing the performance of horticultural species in the field.
Site-specific recombination (SSR) systems have been extensively used for controlled genome modification in eukaryotic cells, especially for DNA excision and DNA site-specific integration. Unidirectional SSR systems are particularly attractive for efficient site-specific gene integration because of their irreversible recombination activities. Bxb1-att is one such unidirectional SSR system, which has emerged as a molecular tool for eukaryotic genome manipulation. We will review the discovery, development, and current applications of the Bxb1-att system with an emphasis on utility in plants. We provide perspectives on future applications using dual integrase cassette exchange for precise gene integration at a specific locus. Finally, potential applications of combining unidirectional SSR systems with newly developed genome-editing tools (e.g., CRISPR-Cas9) will be explored to enhance capabilities for precise locus-specific gene integration into plant genomes.
Site-specific recombination (SSR) systems have been extensively used for controlled genome modification in eukaryotic cells, especially for DNA excision and DNA site-specific integration. Unidirectional SSR systems are particularly attractive for efficient site-specific gene integration because of their irreversible recombination activities. Bxb1-att is one such unidirectional SSR system, which has emerged as a molecular tool for eukaryotic genome manipulation. We will review the discovery, development and current applications of the Bxb1-att system with an emphasis on utility in plants. We provide perspectives on future applications using dual integrase cassette exchange for precise gene integration at a specific locus. Finally, potential applications of combining unidirectional SSR systems with newly developed genome-editing tools (e.g., CRISPR-Cas9) will be explored to enhance capabilities for precise locus-specific gene integration into plant genomes.
Chloroplast is a new hotspot in the field of plant transformation system of plant genetic engineering. Initially developed in Chlamydomonas and tobacco, it is now feasible in a broad range of species. They exploit the homologous recombination and segregation pathways acting on chloroplast genomes and are based on direct repeats, transient co-integration or co-transformation and segregation of trait and marker genes. Foreign site-specific recombinases and their target sites provide an alternative and effective method for removing marker genes from plastids.Chloroplast genetic engineering has many advantages over nuclear genetic enginering, especially site-specific introduction of foreign genes ,leading to the absence of gene siliency and positon effect,which providing the available to explore the regulation and mechanism of chloroplast genes`expression in vitro .It also can identify the structure and function of the chloroplast genome , the expression of chloroplast affect the nulear genome. In this paper, the basic methodology of chloroplast transformation, the current techniques and applications, and the future possibilities for Chloroplast genetic engineering was reviewed[1-3].
During the process of genetic transformation of crop plants with the desired transgene(s), certain selectable marker gene(s) are also employed for the selection of transgenic events. Normally, selective agents like antibiotics and/or herbicides and their corresponding resistant gene(s) are exploited for introducing agronomically important traits/genes into plants. Although these selection genes are imperative for efficient screening, they exhibit regulatory and public concerns. The marker genes present in commercial transgenic crops may be transmitted to pathogenic microbes in the gastrointestinal region/soil or weeds, making them tolerant to antibiotics or herbicides, respectively. Further, sexual breeding of transgenic plants evokes the issue of transgene expression since redundancy of transgenes in the DNA may induce homology-based gene silencing. Therefore, generation of a marker-free transgenic system has become a subject of paramount importance so as to maintain sustainability. Hitherto, various approaches for elimination of marker gene(s) from nuclear as well as chloroplast genomes (co-transformation-segregation, non-selected transformation, site-specific transformation, homologous recombination, transient co-integration) have evolved. In the present chapter, we describe the different marker excision strategies along with their merits and demerits. In addition, we discuss various developments made in marker-free technology and suggest possible directions for their safe and maximum usage in the coming future.
The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.
Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. The example of the marker gene is the barau gene flanked by loxP sites in the plastid genome. For marker excision Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the barau gene, confers a visual aurea leaf phenotype, thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The success of in planta plastid marker excision proves that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA and the availability of visual marker genes.
The sections in this article are
Introduction
Historical Aspects
Unique Features of Chloroplast Genetic Engineering
Maternal Inheritance and Gene Containment
Crop Species Stably Transformed via the Plastid Genome
Agronomic Traits Conferred via the Plastid Genome
Transgenic Plastids as Bioreactors
Biomaterials, Enzymes, and Amino Acids
Conclusions
Acknowledgements
To study the impact of regulatory sequences from Cowpea mosaic virus (CPMV) on Cre-mediated recombination rates, the cre gene was flanked by the 5′ non-translated and 3′ non-translated regions of CPMV. This cre configuration was tested by simultaneous excision of nptII selectable marker gene and heat-inducible cre recombinase gene in potato. Fusion of the cre recombinase sequence with modified regulatory sequences of CPMV increased both the excision efficiencies in primary regenerants and transmission frequencies of recombined loci to vegetative progeny as was confirmed by molecular analysis. These data might have practical implication with regard to selection of putative recombinants in vegetative progeny of potato and other clonally propagated plants as well.
Removal of marker genes improves the design of transgenic plants. Homologous recombination between direct repeats provides a simple method for excising marker genes after transgenic cells and shoots have been isolated. Efficient implementation of the method requires high rates of homologous recombination relative to illegitimate recombination pathways. The procedure works well in plastids where homologous recombination predominates. Marker genes are flanked by engineered direct repeats. The number and length of direct repeats flanking a marker gene influence excision rate. Excision is automatic and loss of the marker gene is controlled by selection alone. After transgenic cells have been isolated selection is removed allowing loss of the marker gene. Excision is a unidirectional process resulting in the rapid accumulation of high levels of marker-free plastid genomes. Cytoplasmic sorting of marker-free plastids from marker-containing plastids leads to the isolation of marker free plants. Marker-free plants can be isolated following vegetative propagation or among the progeny of sexual crosses.
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 studies carried out in the 1950s and 1960s primarily with Chlamydomonas laid the framework for distinguishing plastid and nuclear genomes. Based on this, the coding and regulatory functions of three genomes-nuclear, chloroplast, and mitochondrial, are being addressed in modern plant molecular biology. Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. Chloroplast DNA seems to obey the universal rules. Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein-DNA complexes (called nucleoids). Most plastid genomes are circular and range in size from 120 to 217 kbp. The study of chloroplast gene expression also provides an opportunity to understand how plants sense and alter gene expression in response to light.
Sorghum is essentially grown on marginally fertile land because of its yield stability under adverse conditions with primary distribution in the developing areas of the world, such as Africa and Asia. In the last decade, sorghum (Sorghum bicolor L.) was grown on over 100 million acres each year worldwide and current production is nearly 60 million metric tonnes of grain. It ranks as the sixth most planted crop in the world, behind wheat, rice, corn, soybean and barley. Sorghum was first domesticated as food in East Africa several thousand years ago and is now a dietary staple for over half a billion people in more than 30 countries. It has been used both as an ingredient in food and livestock feed. Like other cereal grains, sorghum’s reputation as either a food or feed crop suffers because of its low protein quality and low content of essential amino acids such as lysine. Imbalanced amino acid composition of cereals, along with low protein content and inadequate energy intake, contribute to protein-energy malnutrition, especially of children, in many countries in Africa and Asia; this affects not only their physical growth, but also their mental development and immune system.
For the engineering of new traits in plants and algae, modification of the plastid genome offers significant advantages. These include transgene integration by homologous recombination, high levels of expression, multigene engineering in polycistronic units, and reduced transmission through pollen. Numerous tools are available for DNA delivery, selection of transformants, removal of undesirable markers and efficient expression of single or multiple transgenes. The list of plants and algae with successful plastid transformation is increasing and includes some crop plants. Its further extension to agronomically relevant species and appropriate cultivars, together with advances in synthetic biology, will be key to the future development of plastids as green factories for the production of high-value metabolites or proteins.
Introduction The Principle of Gene-Deletor Technology and Supporting Experimental Data Characteristics and Requirements of Gene-Deletor Technology Potential Applications of Gene-Deletor Technology Conclusions and Perspectives Acknowledgments References
Selection marker genes (SMGs) have been commonly used in genetic transformation of plants for efficient selection of transformed cells, tissue, or regenerated shoots. In the majority of cases, the selection is based on antibiotic or herbicide resistance. The presence of such genes within the environment or in the food supply might pose an unpredictable hazard to the ecosystem and to human health; therefore research has been initiated to develop an efficient marker-free transgenic system. Various techniques have been developed in recent years to generate marker free transgenic plants and to eliminate marker genes from transgenics. These include site-specific recombination, homologous recombination, transposition, transient co-integration of the marker gene, and a co-transformation-segregation approach, but success has been limited to only a few plant species. Transgenic technology could become more reliable with the improvement of existing marker gene removal strategies and the development of novel approaches for plant genome manipulation. This review describes the contemporary strategies deployed to generate marker-free transgenic plants and marker gene removal, the merits and shortcomings of different approaches, and possible directions for future research programmes.
Since the development of methodologies for achieving genetic modification of chloroplast genomes, termed transplastomic technologies, a straightforward use in the field of plant-based vaccines was initiated. Chloroplast transformation is mediated by homologous recombination that allows for site-specific insertion of foreign DNA into the plastome. This focus offers substantial advantages that include high yields, improved biosafety given by maternal inheritance in most plant species, and multigene expression through polycistrons allowing in theory for the straightforward production of multicomponent vaccines. One limitation of the system consists of the lack of glycosylation pathways, which are of relevance in some cases. State of the art in this area reflects a number of well-characterized vaccination models, although no one has passed clinical evaluations, which contrasts with other nuclear transient expression systems. Transplastomic technologies are envisioned as a relevant tool for developing new convenient vaccines. © 2014 Springer Science+Business Media New York. All rights reserved.
The use of a nonlethal selection scheme, most often using the aadA gene that confers resistance to spectinomycin and streptomycin, has been considered critical for recovery of plastid transformation events. In this study, the plastid-lethal markers, glyphosate or phosphinothricin herbicides, were used to develop a selection scheme for plastids that circumvents the need for integration of an antibiotic resistance marker. The effect of selective agents on tobacco (Nicotiana tabacum) mesophyll chloroplasts was first examined by transmission electron microscopy. We found that at concentrations typically used for selection of nuclear transformants, herbicides caused rapid disintegration of plastid membranes, whereas antibiotics had no apparent effect. To overcome this apparent herbicide lethality to plastids, a “transformation segregation” scheme was developed that used two independent transformation vectors for a cotransformation approach and two different selective agents in a phased selection scheme. One transformation vector carried an antibiotic resistance (aadA) marker used for early nonlethal selection, and the other transformation vector carried the herbicide (CP4 or bar) resistance marker for use in a subsequent lethal selection phase. Because the two markers were carried on separate plasmids and were targeted to different locations on the plastid genome, we reasoned that segregation of the two markers in some transplastomic lines could occur. We report here a plastid cotransformation frequency of 50% to 64%, with a high frequency (20%) of these giving rise to transformation segregants containing exclusively the initially nonselected herbicide resistance marker. Our studies indicate a high degree of persistence of unselected transforming DNA, providing useful insights into plastid chromosome dynamics.
A general method of gene transfer that does not leave behind a selectable marker in the host genome is described. A luciferase gene was introduced into the tobacco genome by using the hygromycin phosphotransferase gene (hpt) as a linked selectable marker. Flanked by recombination sites from the bacteriophage P1 Cre/lox recombination system, the hpt gene was subsequently excised from the plant genome by the Cre recombinase. The Cre-catalyzed excision event in the plant genome was precise and conservative--i.e., without loss or alteration of nucleotides in the recombinant site. After removal of the Cre-encoding locus by genetic segregation, plants were obtained that had incorporated only the desired transgene. Gene transfer without the incorporation of antibiotic-resistance markers in the host genome should ease public concerns over the field release of transgenic organisms expressing such traits. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host.
The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. coli.
Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.
Genetic transformation of plants often results in multiple copies of the introduced DNA at a single locus. To ensure that only a single copy of a foreign gene resides in the plant genome, we used a strategy based on site-specific recombination. The transformation vector consists of a transgene flanked by recombination sites in an inverted orientation. Regardless of the number of copies integrated between the outermost transgenes, recombination between the outermost sites resolves the integrated molecules into a single copy. An example of this strategy has been demonstrated with wheat transformation, where four of four multiple-copy loci were resolved successfully into single-copy transgenes.
The Cre DNA recombinase of bacteriophage P1 has become a useful tool for genomic manipulation in mice and other eukaryotes.
Because Cre is of prokaryotic origin, the 38 kDa protein has been presumed to gain access to the eukaryotic nucleus simply
because it is sufficiently small to pass through the nuclear pore by passive diffusion. Instead, we show here that Cre carries
nuclear targeting determinants that efficiently direct Cre entry into the nucleus of mammalian cells. Fusions of Cre with
green fluorescent protein (GFP) identified two regions that are necessary for nuclear localization. Region I contains a cluster
of basic amino acids that is essential for nuclear localization and which resembles a bipartite-like nuclear localization
signal. Region II exhibits a β-sheet structure with which the bipartite motif may interact. However, neither region is by
itself sufficient for nuclear localization. Nuclear transport in vitro with a 98 kDa GFP-Cre fusion protein shows that Cre does not gain access to the nucleus by passive diffusion, but instead
enters the nucleus by means of an energy-dependent process. Thus, Cre is one of the few prokaryotic proteins that have been
shown to carry determinants that allow it to target the eukaryotic nucleus.
Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.
The bacteriophage P1 Cre/loxP system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice. Although in vitro studies have shown that Cre can catalyze recombination between cryptic "pseudo-loxP" sites in mammalian genomes, to date there have been no reports of loxP-site infidelity in transgenic animals. We produced lines of transgenic mice that use the mouse Protamine 1 (Prm1) gene promoter to express Cre recombinase in postmeiotic spermatids. All male founders and all Cre-bearing male descendents of female founders were sterile; females were unaffected. Sperm counts, sperm motility, and sperm morphology were normal, as was the mating behavior of the transgenic males and the production of two-celled embryos after mating. Mice that expressed similar levels of a derivative transgene that carries an inactive Cre exhibited normal male fertility. Analyses of embryos from matings between sterile Cre-expressing males and wild-type females indicated that Cre-catalyzed chromosome rearrangements in the spermatids that lead to abortive pregnancies with 100% penetrance. Similar Cre-mediated, but loxP-independent, genomic alterations may also occur in somatic tissues that express Cre, but, because of the greater difficulty of assessing deleterious effects of somatic mutations, these may go undetected. This study indicates that, following the use of the Cre/loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.
Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.
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.
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The chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.
Eukaryotic cells are divided into multiple membrane-bound compartments, all of which contain proteins. A large subset of these proteins perform functions that are required in more than one compartment. Although in most cases proteins carrying out the same function in different compartments are encoded by different genes, this is not always true. Numerous examples have now been found where a single gene encodes proteins (or RNAs) found in two (or more) cell organelles or membrane systems. Some particularly clear examples come from protein synthesis itself: plant cells contain three protein-synthesizing compartments, the cytosol, the mitochondrial matrix and the plastid stroma. All three compartments thus require tRNAs and aminoacyl-tRNA synthetases. Some mitochondrial tRNAs and their aminoacyl-tRNA synthetases are identical to their cytosolic counterparts and they are encoded by the same genes. Similarly, some mitochondrial and plastid aminoacyl-tRNA synthetases are encoded by the same nuclear genes. The various ways in which differentially targeted products can be generated from single genes is discussed.
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.
Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.
The transfer of genetic information between distantly or even unrelated organisms during evolution had been inferred from nucleotide sequence comparisons. These studies provided circumstantial evidence that in rare cases genes had been laterally transmitted amongst organisms of the domains bacteria, archaea and eukarya. Laboratory-based studies confirmed that the gene pools of the various domains of organisms are linked. Amongst the bacterial gene exchange mechanisms transduction, transformation and conjugation, the latter was identified as the mechanism with potentially the broadest host range of transfer. Previously, the issue of horizontal gene transfer has become important in the context of biosafety. Gene transfer studies carried out under more natural conditions such as in model ecosystems or in the environment established that all gene transfer mechanisms worked under these conditions. Moreover, environmental hot-spots were identified where favourable conditions such as nutrient enrichment increased the probability of genetic exchange among bacteria. In particular, the phytosphere was shown to provide conducive conditions for conjugative gene exchange. Concern has been expressed that transfer of recombinant DNA (e.g. antibiotic resistance genes) from genetically modified organisms (GMOs) such as transgenic plants to phytosphere bacteria may occur and thus contribute to the undesirable spread of antibiotic resistance determinants. Studies which were performed to address this issue clearly showed that such a transfer occurs, if at all, at extremely low frequency.
The complete sequences of 12 plastid genomes have recently been determined. This has revealed that in addition to known genes involved in plastid gene expression and photosynthesis, there are numerous open reading frames encoding genes of unknown function. Several of these genes have been inactivated in Chlamydomonas reinhardtii and tobacco by biolistic chloroplast transformation. These studies — coupled with molecular, biochemical and biophysical analysis — have revealed the existence of novel subunits and factors required for the accumulation and optimal functioning of photosynthetic complexes. Other plastid genes have also been identified that are essential for cell survival, but whose function remains to be determined.
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.
Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants. In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells. We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein. 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin. The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.
The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction. Lambda infection was used to introduce the cre gene into cells containing plasmid substrates. The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin. Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently. These results are strikingly different from those found with purified enzyme in vitro. Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo. We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.
The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, beta-glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALSr), were integrated into the genome of tobacco and Arabidopsis. The ALSr gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALSr gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The beta-glucuronidase gene remained active. The excision efficiency varied in F1 progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox x Cre F1 progeny were chimeric and some F2 progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.
Cet article de synthese decrit brievement les roles biologiques varies de la recombinaison site-specifique conservative (RSSC) et decrit plus particulierement les proteines et les composantes de l'ADN des systemes RSSC et les mecanismes de recombinaison
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 report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.
We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICE1 sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the ColE1 ori for maintenance in E. coli and a spectinomycin resistance gene (aadA) for selection in both systems. In addition, pNICER1 carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICER1 was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.
The bacterial gene aadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of the aadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking the aadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which the aadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas the aadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse the aadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.
We have determined the complete sequence of the mitochondrial DNA in the model plant species Arabidopsis thaliana, affording access to the first of its three genomes. The 366,924 nucleotides code for 57 identified genes, which cover only 10% of the genome. Introns in these genes add about 8%, open reading frames larger than 100 amino acids represent 10% of the genome, duplications account for 7%, remnants of retrotransposons of nuclear origin contribute 4% and integrated plastid sequences amount to 1%-leaving 60% of the genome unaccounted for. With the significant contribution of duplications, imported foreign DNA and the extensive background of apparently functionless sequences, the mosaic structure of the Arabidopsis thaliana mitochondrial genome features many aspects of size-relaxed nuclear genomes.
During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites. The bacteriophage recombinase Cre catalyses site-specific recombination between two 34-base-pair loxP sites. The crystal structure at 2.4 A resolution of Cre bound to a loxP substrate reveals an intermediate in the recombination reaction, in which a Cre molecule has cleaved the substrate to form a covalent 3'-phosphotyrosine linkage with the DNA. Four recombinases and two loxP sites form a synapsed structure in which the DNA resembles models of four-way Holliday-Junction intermediates. The Cre-loxP complex challenges models of site-specific recombination that require large changes in quaternary structure. Subtle allosteric changes at the carboxy termini of the Cre subunits may instead coordinate the cleavage and strand-exchange reactions.
The enzyme cytosine deaminase (CD) encoded by codA catalyzes deamination of cytosine to uracil. CD is present in prokaryotes and in many eukaryotic micro-organisms, but is absent in higher plants. 5-fluorocytosine (5FC) is metabolized in CD-expressing cells, causing cellular death. A chimeric codA has been introduced into the tobacco plastid genome and 5FC was used to select against tissue culture cells and seedlings expressing CD. This negative selection scheme will be useful in identifying nuclear genes which control plastid gene expression in higher plants.
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.
The frequency of possible "horizontal" gene transfer between a plant and a tightly associated bacterial pathogen was studied in a model system consisting of transgenic Solanum tuberosum, containing a beta-lactamase gene linked to a pBR322 origin of replication, and Erwinia chrysanthemi. This experimental system offers optimal conditions for the detection of possible horizontal gene transfer events, even when they occur at very low frequency. Horizontal gene transfer was not detected under conditions mimicking a "natural" infection. The gradual, stepwise alteration of artificial, positive control conditions to idealized natural conditions, however, allowed the characterization of factors that affected gene transfer, and revealed a gradual decrease of the gene transfer frequency from 6.3 x 10(-2) under optimal control conditions to a calculated 2.0 x 10(-17) under idealized natural conditions. These data, in combination with other published studies, argue that horizontal gene transfer is so rare as to be essentially irrelevant to any realistic assessment of the risk involved in release experiments involving transgenic plants.
The chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.
Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally. Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes. The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich. Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T(0) transplastomic plants containing the bar gene. Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T(2) plants containing a plastid-located uidA reporter gene.
Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.
Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.
The two most abundant transcripts derived from TR-DNA within plant cells transformed by an octopine strain of Agrobacterium tumefaciens arise from divergent transcription, both originating within an 500 bp section of the T-DNA. Using a combination of subcloning and exonuclease digestion, a 479-bp DNA fragment, directly flanked by the initiation codons for the two adjacent open reading frames, was isolated. The resulting DNA fragment was fused, in both orientations, to the neomycin phosphotransferase (NPT II) gene of the transposon Tn5 prior to introduction into Nicotiana tabacum cells via the Ti plasmid. The intergenic fragment was found to initiate expression of the NPT II gene in either orientation as assayed by kanamycin resistance of the transformed plant tissue as well as by enzymatic assay of the NPT II gene product. The plasmids described here are potential selection-expression vectors for plant systems.
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