The salt-tolerance gene rstB can be used as a selectable marker in plant genetic transformation

The salt-tolerance gene rstB under the control of the cauliflower mosaic virus 35S promoter was used as a selectable marker gene in the Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum cv. Xanthi). The selective agent for plant regeneration was tolerance to 170 mM sodium chloride. The highest selection efficiency was 83.3%. No obvious differences in selection efficiencies were observed when those obtained using the standard selectable marker gene hpt and a selection regime of 10 mg l⁻¹ hygromycin. Transgenic events were confirmed by PCR, Southern blot, RT-PCR and green fluorescent protein studies. The rstB transgenic plants showed improved salt tolerance and a normal phenotype. Based on these results, we suggest that the rstB gene may be used as a promising selectable marker and an alternative to the antibiotic- or herbicide-resistance genes in plant transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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     

The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker

We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.     

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.     

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T₁ soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.     

Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids

A phytoene desaturase (PDS) gene was cloned and characterized from the unicellular green microalga Chlamydomonas reinhardtii. Functional complementation analysis revealed C. reinhardtii PDS (CrPDS) catalyzes the conversion of phytoene to the colored carotenoid ζ-carotene. A single amino acid substitution, L505F, enhanced its desaturation activity by 29%, as indicated by an in vitro enzymatic assay. In addition, CrPDS-L505F exhibited 27.7-fold higher resistance to the herbicide norflurazon. Glass bead-mediated delivery displayed a high transformation efficiency of C. reinhardtii with CrPDS-L505F, demonstrating clearly that the engineered endogenous CrPDS is a dominant selectable marker for C. reinhardtii and possibly for other green algae. Furthermore, the expression of PDS could enhance the intracellular carotenoid accumulation of transformants, opening up the possibility of engineering the carotenogenic pathway for improved carotenoid production in microalgae.     

Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics

Selectable marker gene systems are vital for the development of transgenic crops. Since the creation of the first transgenic plants in the early 1980s and their subsequent commercialization worldwide over almost an entire decade, antibiotic and herbicide resistance selectable marker gene systems have been an integral feature of plant genetic modification. Without them, creating transgenic crops is not feasible on purely economic and practical terms. These systems allow the relatively straightforward identification and selection of plants that have stably incorporated not only the marker genes but also genes of interest, for example herbicide tolerance and pest resistance. Bacterial antibiotic resistance genes are also crucial in molecular biology manipulations in the laboratory. An unprecedented debate has accompanied the development and commercialization of transgenic crops. Divergent policies and their implementation in the European Union on one hand and the rest of the world on the other (industrialized and developing countries alike), have resulted in disputes with serious consequences on agricultural policy, world trade and food security. A lot of research effort has been directed towards the development of marker-free transformation or systems to remove selectable markers. Such research has been in a large part motivated by perceived problems with antibiotic resistance selectable markers; however, it is not justified from a safety point of view. The aim of this review is to discuss in some detail the currently available scientific evidence that overwhelmingly argues for the safety of these marker gene systems. Our conclusion, supported by numerous studies, most of which are commissioned by some of the very parties that have taken a position against the use of antibiotic selectable marker gene systems, is that there is no scientific basis to argue against the use and presence of selectable marker genes as a class in transgenic plants.     

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.     

Effect of promoter driving selectable marker on corn transformation

Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.     

A point mutation in the Medicago sativa GSA gene provides a novel, efficient, selectable marker for plant genetic engineering

Bacterial selectable marker genes (SMG) conferring antibiotic resistance are valuable tools in plant genetic engineering, but public concern and regulatory requirements have stimulated the development of alternative selection systems. We have previously demonstrated that a mutated Synechococcus elongatus HemL gene encoding glutamate 1-semialdehyde aminotransferase (GSA) is an efficient SMG in alfalfa. In fact, GSA is irreversibly inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid), but the mutated enzyme is gabaculine insensitive. With the aim to develop a plant derived SMG, we cloned and sequenced the Medicago sativa GSA cDNA and reproduced one of the two mutations associated with gabaculine resistance in Synechococcus, a transversion resulting in a methionine to isoleucine (M → I) substitution. This mutated gene was assessed as a SMG in tobacco and alfalfa Agrobacterium transformation, in comparison with the wild type gene. In tobacco, about 43% of the leaf explants produced green shoots, whereas in alfalfa 47% of the explants produced green embryos in the presence of 30 μM gabaculine when the M → I GSA was introduced. Escapes were absent in tobacco and only 6% in alfalfa. No effect on the plant phenotype was noticed. We propose this new SMG as a widely acceptable alternative to those currently used.     

Sulfonamide resistance gene for plant transformation

The sulfonamide resistance gene from plasmid R46 encodes for a mutated dihydropteroate synthase insensitive to inhibition by sulfonamides. Its coding sequence was fused to the pea ribulose bisphosphate carboxylase/oxygenase transit peptide sequence. Incubation of isolated chloroplasts with the fusion protein synthesised in vitro, showed that the bacterial enzyme was transported to the chloroplast stroma and processed into a mature form. Expression of the gene fusion in transgenic plants resulted in a high level of resistance to sulfonamides. Direct selection of transformed shoots on leaf explants was efficient using sulfonamides as sole selective agents. Transformed shoots rooted normally on sulfonamides at concentrations toxic for untransformed ones. Sulfonamide resistance was transmitted to the progeny of transformed plants as a single Mendelian dominant character. These results demonstrate that this chimeric gene can be used as an efficient and versatile selectable marker for plant transformation.     

Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection

This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

Generation of transgenic asulam-resistant potatoes to facilitate eradication of parasitic broomrapes (Orobanche spp.), with the sul gene as the selectable marker

 Root-attaching parasitic flowering broomrapes (Orobanche spp.) are major constraints to vegetable, legume and sunflower production around the Mediterranean and elsewhere, with banned methyl-bromide fumigation or land abandonment of these affected crops as the major “solutions” to the problem. We report the specific generation of transgenic asulam-resistant potatoes as a way to eradicate this pest. The target-site resistance in the crop allows the herbicide to be translocated from treated leaves to the parasite via crop roots. This inhibitor of dihydropteroate synthase lethally then prevents folic acid biosynthesis in the parasite. Additionally, we demonstrate that asulam can be used directly in selecting resistant transformants, without the need for another selectable marker. Received: 18 June 1997 / Accepted: 17 July 1997     

A chimaeric hygromycin resistance gene as a selectable marker in plant cells

Summary We show here that plant cells are sensitive to the antibiotic hygromycin-B⁴. We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.     

Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L. cv. Zhongmian 35) using glyphosate as a selectable marker

The most economically significant Chinese cotton cultivar (Gossypium hirsutum L. cv. Zhongmian 35) was transformed via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast-transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium containing glyphosate. Thirty-four independent transgenic lines were obtained after selection, giving a maximal 1.9% transformation frequency. The integration and expression of the aroA-M1 gene in T₀ plants and T₁ progeny were confirmed using DNA hybridization, Western blot and PCR techniques. An increased resistance of T₀ and T₁ transgenic plants towards glyphosate was also observed.     

The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri

The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3′-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker – paromomycin resistance (Pm^R) – for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3′ UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable Pm^R transformants ranged about 15 per 10⁶ target cells. Due to rapid and sharp selection, Pm^R transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of Pm^R transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.     

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene

Positive selection of transgenic plants is essential during plant transformation. Thus, strong promoters are often used in selectable marker genes to ensure successful selection. Many plant transformation vectors, including pPZP family vectors, use the 35S promoter as a regulatory sequence for their selectable marker genes. We found that the 35S promoter used in a selectable marker gene affected the expression pattern of a transgene, possibly leading to a misinterpretation of the result obtained from transgenic plants. It is likely that the 35S enhancer sequence in the 35S promoter is responsible for the interference, as in the activation tagging screen. This affected expression mostly disappeared in transgenic plants generated using vectors without the 35S sequences within their T-DNA region. Therefore, we suggest that caution should be used in selecting a plant transformation vector and in the interpretation of the results obtained from transgenic approaches using vectors carrying the 35S promoter sequences within their T-DNA regions.