Generation of marker-free plastid transformants using a transiently cointegrated selection gene

Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.     

Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation

We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aad A-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atp B promoter region. Efficient recombination between atp B repeats deletes the intervening foreign genes and 1984 bp of plastid DNA (co-ordinates 57 424–59 317) containing the rbc L gene. Only five foreign bases are present in Δ rbc L plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbc L-related sequences in Δ rbc L plants indicated an extra-plastidic origin. Mutant Δ rbc L plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aad A-based transformation with the wild-type rbc L gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aad A genes. Rescue of the Δ rbc L mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbc L deletion mutant isolated here is readily transformed with the efficient aad A marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.     

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.

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.     

Efficient plastid transformation in tobacco using the<Emphasis Type="Italic"> aphA-6</Emphasis> gene and kanamycin selection

Here we report on the development of a new dominant selection marker for plastid transformation in higher plants using the aminoglycoside phosphotransferase gene aphA-6 from Acinetobacter baumannii. Vectors containing chimeric aphA-6 gene constructs were introduced into the tobacco chloroplast using particle bombardment of alginate-embedded protoplast-derived micro colonies or polyethylene glycol (PEG)-mediated DNA uptake. Targeted insertion into the plastome was achieved via homologous recombination, and plastid transformants were recovered on the basis of their resistance to kanamycin. Variations in kanamycin resistance in transplastomic lines were observed depending on the 5' and 3' regulatory elements associated with the aphA-6 coding region. Transplastomic plants were fertile and showed maternal inheritance of the transplastome in the progeny.     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

Chloramphenicol acetyltransferase as selectable marker for plastid transformation

Chloroplast transformation remains a demanding technique and is still restricted to relatively few plant species. The limited availability of selectable marker genes and the lack of selection markers that would be universally applicable to all plant species represent some of the most serious technical problems involved in extending the species range of plastid transformation. Here we report the development of the chloramphenicol acetyltransferase gene cat as a new selectable marker for plastid transformation. We show that, by selecting for chloramphenicol resistance, tobacco chloroplast transformants are readily obtained. Transplastomic lines quickly reach the homoplasmic state (typically in one additional regeneration round), accumulate the chloramphenicol acetyltransferase enzyme to high levels and transmit their plastid transgenes maternally into the next generation. No spontaneous antibiotic resistance mutants appear upon chloramphenicol selection. Several lines of evidence support the assumption that plant mitochondria are also sensitive to chloramphenicol suggesting that the chloramphenicol acetyltransferase may be a good candidate selectable marker for plant mitochondrial transformation.     

The chloroplast transformation toolbox: selectable markers and marker removal

Plastid transformation is widely used in basic research and for biotechnological applications. Initially developed in Chlamydomonas and tobacco, it is now feasible in a broad range of species. Selection of transgenic lines where all copies of the polyploid plastid genome are transformed requires efficient markers. A number of traits have been used for selection such as photoautotrophy, resistance to antibiotics and tolerance to herbicides or to other metabolic inhibitors. Restoration of photosynthesis is an effective primary selection method in Chlamydomonas but can only serve as a screening tool in flowering plants. The most successful and widely used markers are derived from bacterial genes that inactivate antibiotics, such as aadA that confers resistance to spectinomycin and streptomycin. For many applications, the presence of a selectable marker that confers antibiotic resistance is not desirable. Efficient marker removal methods are a major attraction of the plastid engineering tool kit. 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.     

Removal of antibiotic resistance genes from transgenic tobacco plastids

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.     

Combining a regeneration-promoting <Emphasis Type="Italic">ipt</Emphasis> gene and site-specific recombination allows a more efficient apricot transformation and the elimination of marker genes

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In addition, their elimination may allow gene stacking by the same selection strategy. In apricot, selection using the selectable marker gene nptII, that confers resistance to aminoglycoside antibiotics, is relatively effective. An attractive alternative is offered by the MAT system (multi-auto-transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with an MAT vector has been attempted in the apricot cultivar ‘Helena’. Regeneration from infected leaves with Agrobacterium harboring a plasmid containing the ipt gene was significantly higher than that from non-transformed controls in a non-selective medium. In addition, transformation efficiencies were much higher than those previously reported using antibiotic selection, probably due to the integration of the regeneration-promoting ipt gene. However, the lack of an ipt expression-induced differential phenotype in apricot made difficult in detecting the marker genes excision and plants had to be evaluated at different times. PCR analysis showed that cassette excision start occurring after 6 months approximately and 1 year in culture was necessary for complete elimination of the cassette in all the transgenic lines. Excision was confirmed by Southern blot analysis. We report here for the first time in a temperate fruit tree that the MAT vector system improves regeneration and transformation efficiency and would allow complete elimination of marker genes from transgenic apricot plants by site-specific recombination.     

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.     

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas. Received: 26 October 1999 / Accepted: 28 December 1999     

A novel approach to plastid transformation utilizes the phiC31 phage integrase

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.     

The maize CorA/MRS2/MGT-type Mg transporter,ZmMGT10, responses to magnesium deficiency and confers low magnesium tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Key message

A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants.

Abstract

More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6′)-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.

     

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.     

Modifying fatty acid profiles through a new cytokinin‐based plastid transformation system

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin‐based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3‐ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over‐expression of isopentenyl transferase. Over‐expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic‐based selection systems, and may be used in combination with a negative selectable marker as part of a two‐step selection system to obtain homoplasmic plant lines.     

<Emphasis Type="Italic">AtMYB12</Emphasis> gene: a novel visible marker for wheat transformation

Efficiency of plant transformation is less than optimal for many important species, especially for monocots which are traditionally recalcitrant to transformation, such as wheat. And due to limited number of selectable marker genes, identification or selection of those cells that have integrated DNA into appropriate plant genome and to regenerate fully developed plants from the transformed cells, becomes even more difficult. Some of the widely used marker genes belong to the categories of herbicide or antibiotic resistance genes and flourescent protein genes. As they become an integral part of plant genome along with promoters prokaryotic or eukaryotic origin, there are certain health and environmental concerns about the use of these reporter genes. These marker genes are also inefficient with respect to time and space. In this study we have found a novel visible selection agent AtMYB12, to screen transgenic wheat, with in days after transformation. Transformed coleoptiles as well as cells regenerating from transformed cultured scutella, phenotypically exhibit purple pigmentation, making selection possible in limited and reasonable cost, time and space.     

New connections across pathways and cellular processes: industrialized mutant screening reveals novel associations between diverse phenotypes in Arabidopsis

In traditional mutant screening approaches, genetic variants are tested for one or a small number of phenotypes. Once bona fide variants are identified, they are typically subjected to a limited number of secondary phenotypic screens. Although this approach is excellent at finding genes involved in specific biological processes, the lack of wide and systematic interrogation of phenotype limits the ability to detect broader syndromes and connections between genes and phenotypes. It could also prevent detection of the primary phenotype of a mutant. As part of a systems biology approach to understand plastid function, large numbers of Arabidopsis thaliana homozygous T-DNA lines are being screened with parallel morphological, physiological, and chemical phenotypic assays (www.plastid.msu.edu). To refine our approaches and validate the use of this high-throughput screening approach for understanding gene function and functional networks, approximately 100 wild-type plants and 13 known mutants representing a variety of phenotypes were analyzed by a broad range of assays including metabolite profiling, morphological analysis, and chlorophyll fluorescence kinetics. Data analysis using a variety of statistical approaches showed that such industrial approaches can reliably identify plant mutant phenotypes. More significantly, the study uncovered previously unreported phenotypes for these well-characterized mutants and unexpected associations between different physiological processes, demonstrating that this approach has strong advantages over traditional mutant screening approaches. Analysis of wild-type plants revealed hundreds of statistically robust phenotypic correlations, including metabolites that are not known to share direct biosynthetic origins, raising the possibility that these metabolic pathways have closer relationships than is commonly suspected.     

2-Deoxyglucose resistance: a novel selection marker for plant transformation

A novel selection marker for plant transformation alternative to antibiotic and herbicide resistance is described. The selective agent applied is 2-deoxyglucose (2-DOG) which in the cytosol of plant cells is phosphorylated by hexokinase yielding 2-DOG-6-phosphate (2-DOG-6-P). 2-DOG-6-P exerts toxic effects on overall cellular metabolism leading to cell death. We observed that constitutive expression of the yeast DOG ^R1 gene encoding a 2-DOG-6-P phosphatase resulted in resistance towards 2-DOG in transgenic tobacco plants. This finding was exploited to develop a selection system during transformation of tobacco and potato plants. The lowest concentration of 2-DOG leading to nearly complete inhibition of regeneration of wild-type explants was found to range between 400 and 600 mg/l 2-DOG for tobacco, potato and tomato plants. After Agrobacterium tumefaciens-mediated transformation cells expressing the DOG ^R1 gene were selected by resistance to 2-DOG. More than 50% of tobacco explants formed shoots and on average 50% of these shoots harboured the DOG ^R1 gene. Similar results were obtained for potato cv. Solara. The acceptability of the resistance gene derived from baker's yeast, the unobjectionable toxicological data of 2-DOG as well as the normal phenotype of DOG ^R1-expressing plants support the use of this selection system in crop plant transformation.     

Utilization of the plant methionine sulfoxide reductase B genes as selectable markers in Arabidopsis and tomato transformation

Plant transformation is an important tool for basic research and agricultural biotechnology. In most cases, selection of putative transformants is based on antibiotic or herbicide resistance. Overexpression of plant genes that provide protection from abiotic or biotic stresses can result in a conferred phenotype that can be used as a means for selection. We have demonstrated herein that specific methionine sulfoxide reductase B (MsrB) genes that are overexpressed in transgenic plants may constitute a new selectable marker with concomitantly increased tolerance to methyl viologen (MV) treatment. Arabidopsis transformants overexpressing cytosolic MsrB7, MsrB8 or MsrB9 are viable and survive after MV selection. To establish whether these native plant origin genes serve as new non-antibiotic markers that can be applied to crop transformation, tomato cotyledons were used as transformation materials. MsrB7 transgenic tomato plants were successfully obtained by Agrobacterium-mediated transformation and selection on medium supplemented with MV. We suggest that specific MsrB genes that are overexpressed in transgenic plants may constitute a new selectable marker with increased tolerance to oxidative stress concomitant with MV treatment.