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.     

<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.     

Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Inducible Excision of Selectable Marker Gene from Transgenic Plants by the Cre/<Emphasis Type="Italic">lox</Emphasis> Site-specific Recombination System

In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.     

转基因植物中标记基因的剔除

在目前的植物转化系统中,要求在关注基因或目的基因转入细胞时,同时有标记基因存在.标记基因主要是抗生素或除草剂的抗性基因.借标记基因的表达可以将转化细胞从大量的未转化细胞中筛选出来,但标记基因的继续存在,特别是在转基因食品中,是人们广泛关注的问题.培育无标记基因的转基因植株已成为植物生物工程研究中的新课题.该文介绍了剔除标记基因的两种方法:分离剔除和重组剔除,并对近年来这两种方法在培育无标记基因的转基因植物中的应用和进展作了介绍.     

A high‐throughput transformation system allows the regeneration of marker‐free plum plants (Prunus domestica)

A high‐throughput transformation system previously developed in our laboratory was used for the regeneration of transgenic plum plants without the use of antibiotic selection. The system was first tested with two experimental constructs, pGA482GGi and pCAMBIAgfp94(35S) that contain selective marker and reporter genes. Transformation was monitored by GUS detection, and estimated transformation efficiencies were 5.7% and 17.7% for pGA482GGi and pCAMBIAgfp94(35S), respectively. Subsequently, an intron‐hairpin‐RNA (ihpRNA) construct, carrying the Plum Pox Virus coat protein (ppv‐cp) gene, without selectable or reporter marker genes was designed. Five transgenic lines were regenerated as confirmed by DNA blot analysis. We believe that this is the first report on the production of marker‐free plants transformed with a potential agronomically important trait in a Prunus species.     

Advances in selectable marker genes for plant transformation

Plant transformation systems for creating transgenics require separate process for introducing cloned DNA into living plant cells. Identification or selection of those cells that have integrated DNA into appropriate plant genome is a vital step to regenerate fully developed plants from the transformed cells. Selectable marker genes are pivotal for the development of plant transformation technologies because marker genes allow researchers to identify or isolate the cells that are expressing the cloned DNA, to monitor and select the transformed progeny. As only a very small portion of cells are transformed in most experiments, the chances of recovering transgenic lines without selection are usually low. Since the selectable marker gene is expected to function in a range of cell types it is usually constructed as a chimeric gene using regulatory sequences that ensure constitutive expression throughout the plant. Advent of recombinant DNA technology and progress in plant molecular biology had led to a desire to introduce several genes into single transgenic plant line, necessitating the development of various types of selectable markers. This review article describes the developments made in the recent past on plant transformation systems using different selection methods adding a note on their importance as marker genes in transgenic crop plants.     

Genetic transformation of apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis>) without use of a selectable marker gene

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition, and cotransformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations for the public. With this goal in mind, a technique for apple transformation was developed without use of any selectable marker. Transformation of the apple genotype “M.26” with the constructs pPin2Att35SGUSintron and pPin2MpNPR1 was achieved. In different experiments, 22.0–25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of the lines are uniformly transformed plants, although some lines are chimeric, as also occurs with the conventional transformation procedure using a selectable marker gene. A second genotype of apple, “Galaxy,” was also transformed with the same constructs, with a transformation efficiency of 13%.     

Effective selection of transgenic papaya plants with the PMI/Man selection system

The selectable marker gene phospho-mannose isomerase (pmi), which encodes the enzyme phospho-mannose isomerase (PMI) to enable selection of transformed cell lines on media containing mannose (Man), was evaluated for genetic transformation of papaya (Carica papaya L.). We found that papaya embryogenic calli have little or no PMI activity and cannot utilize Man as a carbon source; however, when calli were transformed with a pmi gene, the PMI activity was greatly increased and they could utilize Man as efficiently as sucrose. Plants regenerated from selected callus lines also exhibited PMI activity but at a lower specific activity level. Our transformation efficiency with Man selection was higher than that reported using antibiotic selection or with a visual marker. For papaya, the PMI/Man selection system for producing transgenic plants is a highly efficient addition to previously published methods for selection and may facilitate the stacking of multiple transgenes of interest. Additionally, since the PMI/Man selection system does not involve antibiotic or herbicide resistance genes, its use might reduce environmental concerns about the potential flow of those genes into related plant populations.     

Positive selectable marker genes for routine plant transformation

Summary Plant genetic transformation technologies rely upon the selection and recovery of transformed cells. Selectable marker genes used so far have been either antibiotic resistance genes or herbicide tolerance genes. There is a need to apply alternative principles of selection, as more transgenic traits have to be incorporated into a transgenic crop and because of concern that the use of conventional marker genes may pose a threat to humans and the environment. New classes of marker genes are now available, conferring metabolic advantage of the transgenic cells over the non-transformed cells. The new selection systems, as described in this review, are being used with success and superior performance over the traditional marker systems.     

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

A simple method for the production of highly competent cells ofAgrobacterium for transformation via electroporation

The introduction of binary plasmids intoAgrobacterium hosts forAgrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations ofAgrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformedAgrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of threeArgobacterium tumefaciens (EHA101, LBA4404, C58) and twoArgobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin^res, kanamycin^res, and gentamycin^res).     

Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis

Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistance genes such as aadA, nptII and aphA-6. An improvement would be the combination of such a marker with a visual screening system for the early and conclusive detection of plastid transformants. As such, we investigated the use of three photosynthesis-deficient plastid mutants, DeltapetA, Deltaycf3 and DeltarpoA, for the development of a phenotypic selection system. Mutant plants were used as an alternative to the wild-type as source tissue for transformation, re-introducing deleted plastid sequences and using the aphA-6 gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted in shoots with a visually distinct phenotype comparable to the wild-type. This transformation/selection system overcomes the common problems associated with plastid transformation, e.g. the recovery of spontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection, phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstituted plants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of an antibiotic selection agent are no longer required.     

Transformation of suspension cultures of bromegrass (Bromus inermis) by Agrobacterium tumefaciens

Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS) and green fluorescent protein (GFP) genes were co-cultivated for 3 days with bromegrass cells at the late exponential or early stationary growth phase (7–9 days after subculture). These conditions gave optimal transformation efficiency. Putative transformants were identified by selection for geneticin resistance and by examining the calluses using fluorescence microscopy. This allows the elimination of escapes and selection of cells that express the target genes. PCR and Southern blot analyses confirmed the integration of the GUS and GFP genes into the genome of transformed bromegrass cell lines. Transformants with various levels of GUS expression were obtained with a high frequency following Agrobacterium-mediated gene transfer and visual selection by GFP. The successful transformation method described allows reverse genetics approaches for analyzing cold hardiness genes isolated from bromegrass cells.     

A novel robust heat-inducible promoter for heterologous gene expression in Tetrahymena thermophila

An increasing amount of data has revealed the importance of inducible promoters in ciliate research and in ciliate-related industries. However, knowledge about these promoters and related genes is relatively sparse. Here we report a novel inducible promoter from a Tetrahymena cytoplasmic Hsp70 gene member, HSP70-2. The reported promoter was able to induce the endogenous gene up to ～9000-fold after a short heat shock treatment and this remarkable feature has been retained when a relatively short region of the promoter was introduced into a reporter construct followed by transformation. During the recovery period following a short heat shock, both the mRNA and protein levels of the reporter gene were maintained high up to two hours. A constant heat shock treatment to the transformed cells led to a stabilization of the reporter mRNA up to at least six hours and the reporter protein continued to accumulate up to around three hours. The promoter strength appears to be similar to that of the cadmium-induced metallothionein gene (MTT1) promoter. Therefore, the HSP70-2 promoter represents an attractive alternative for the over-expression of proteins in Tetrahymena, and the promoter-reporter gene construct used in this study is an ideal tool to help in understanding the regulation mechanisms of heat shock genes in ciliates.