Feasibility of the seed specific cruciferin C promoter in the self excision Cre/<Emphasis Type="Italic">lox</Emphasis>P strategy focused on generation of marker-free transgenic plants

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T₀ plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T₀ transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T₀-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T₂ seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T₁ plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Application of Arabidopsis tissue-specific CRUC promoter in the Cre/loxP self-excision strategy for generation of marker-free oilseed rape: potential advantages and drawbacks

The aim of the present study was to explore the usability of Arabidopsis cruciferin C (CRUC) promoter in the Cre/loxP self-excision strategy with the minimal rate of an ectopic expression of the cre recombinase. For this, a plant transformation vector containing the gus reporter gene driven by the light-sensitive Lhca3.St1 instead of double dCaMV 35S promoter and one pair loxP sites flanking the cre and the nptII genes was prepared. Agrobacterium-mediated transformations of three economically important oilseed rape (Brassica napus L.) cultivars Campino, Hunter and Topas as well as tobacco (Nicotiana tabacum L.) as a reference system were performed. The integration of the T-DNA into genome of all Brassica cultivars was accompanied by DNA deletions/rearrangements on the both T-DNA ends. The disruption of the Cre/loxP recombination system in oilseed rape was observed. On the contrary, no such events were detected in tobacco. The applied strategy did restrict an ectopic CRUC activity to some extent and a set of transgenic tobacco T₀ plants without premature excision event was obtained. Our data showed that a choice of the promoter can limit undesired activation of the Cre/loxP cassette.     

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Cre/<Emphasis Type="Italic">lox</Emphasis>-mediated marker gene excision in transgenic maize (Zea mays L.) plants

After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. We have tested the Cre/lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants. Two strategies, crossing and autoexcision, have been tested and demonstrated. In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites. Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F₁ plants from most crosses with multiple independent Cre and lox lines. In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene. Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes. Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable. The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.Communicated by D. Hoisington     

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T₀ plants with ASAL- lox-hpt-lox T-DNA and three single-copy T₀ plants with cre-bar T-DNA. Marker gene excisions were detected in T₁ hybrids through hygromycin sensitivity assay. Molecular analysis of T₁ plants exhibited 27.4% recombination efficiency. T₂ progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T₂ progeny plants. In planta bioassay of GLH and BPH performed on these T₂ progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.     

Marker Assisted Transfer of Two Powdery Mildew Resistance Genes PmTb7A.1 and PmTb7A.2 from Triticum boeoticum (Boiss.) to Triticum aestivum (L.)

Powdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the important wheat diseases, worldwide. Two PM resistance genes, designated as PmTb7A.1 and PmTb7A.2, were identified in T. boeoticum acc. pau5088 and mapped on chromosome 7AL approximately 48cM apart. Two resistance gene analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were identified to be linked to the PmTb7A.1 and PmTb7A.2, at a distance of 0.6cM and 6.0cM, respectively. In the present study, following marker assisted selection (MAS), the two genes were transferred to T. aestivum using T. durum as bridging species. As many as 12,317 florets of F₁ of the cross T. durum /T. boeoticum were pollinated with T. aestivum lines PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively, of three-way F₁. The resulting F₁s of the cross T. durum/T. boeoticum//T. aestivum were screened with marker flanking both the PM resistance genes PmTb7A.1 and PmTb7A.2 (foreground selection) and the selected plants were backcrossed to generate BC₁F₁. Marker assisted selection was carried both in BC₁F₁ and the BC₂F₁ generations. Introgression of alien chromatin in BC₂F₁ plants varied from 15.4 - 62.9 percent. Out of more than 110 BC₂F₁ plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants with minimum of the alien introgression. Cytological analysis showed that most plants have chromosome number ranging from 40-42. The BC₂F₂ plants homozygous for the two genes have been identified. These will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien introgression. The PM resistance gene PmTb7A.1 maps in a region very close to Sr22, a stem rust resistance gene effective against the race Ug99. Analysis of selected plants with markers linked to Sr22 showed introgression of Sr22 from T. boeoticum in several BC₂F₁ plants. Thus, in addition to PM resistance, these progeny might also carry resistance to stem rust race Ug99.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Evaluation of codA,tms2, and ABRIN-A as negative selectable markers in transgenic tobacco and rice

The codA and tms2 genes are used as efficient conditional negative selectable markers (NSMs) in several dicotyledonous plants. We evaluated both genes under control of the CaMV 35S promoter for their effectiveness as conditional NSMs. The ABRIN-A chain gene from Abrus precatorius was evaluated as a nonconditional NSM. The efficacies of codA, tms2, and ABRIN-A as NSMs were compared in transgenic rice and tobacco. Tobacco leaf discs and scutellum-derived callus of rice were transformed with the three genes. Leaf discs of T₀ transgenic tobacco plants and the T₁ seedlings of transgenic rice plants, both transformed with codA, showed a pronounced reduction in growth in the presence of the substrate 5-fluorocytosine. The tms2 gene was inferred to act as a nonconditional NSM in tobacco since all the recovered hygromycin-resistant transgenic tobacco plants harbored only truncated transferred DNAs (T-DNAs) with deletions of the tms2 gene. The T₁ transgenic rice seedlings transformed with tms2 showed a drastic reduction in shoot and root growth in the presence of the substrate naphthaleneacetamide. Both codA and tms2 genes served as good conditional NSMs in rice. The ABRIN-A gene proved to be a good nonconditional NSM in tobacco since all recovered hygromycin-resistant plants harbored only truncated T-DNAs with deletions of the ABRIN-A gene. Twelve transgenic rice plants, which harbored the complete ABRIN-A gene, displayed normal growth suggesting that ABRIN-A is not toxic to rice.     

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.     

Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites

The Cre-lox system is an important tool for genetic manipulation. To promote integrative reactions, two strategies using mutant lox sites have been developed. One is the left element/right element (LE/RE)-mutant strategy and the other is the cassette exchange strategy using heterospecific lox sites such as lox511 or lox2272. We compared the recombination efficiencies using these mutant lox sites in embryonic stem (ES) cells, and found that the combination of the LE/RE mutant and lox2272 showed high recombination efficiency and stability of the recombined structure. Taking advantage of this stability, we successfully integrated the cre gene into the mutant lox sites by Cre-mediated recombination. Germ line chimeric mice were produced from the cre-integrated ES cell clones, and Cre-expressing mouse lines were established. The inserted cre gene was stably maintained through the generations. This cre knock-in system using the LE/RE-lox2272 combination should be useful for the production of various cre mice for gene targeting or gene trapping.     

Cre/ lox site-specific recombination controls the excision of a transgene from the rice genome

The Cre/lox site-specific recombination controls the excision of a target DNA segment by recombination between two lox sites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase (hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre/lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre/lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre/lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS⁺ plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages. Received. 2 April 2001 / Accepted: 29 June 2001     

Type I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.)

To develop a less genotype-dependent maize-transformation procedure, we used 10-month-old Type I callus as target tissue for microprojectile bombardment. Twelve transgenic callus lines were obtained from two of the three anther-culture-derived callus cultures representing different gentic backgrounds. Multiple fertile transgenic plants (T₀) were regenerated from each transgenic callus line. Transgenic leaves treated with the herbicide Basta showed no symptoms, indicating that one of the two introduced genes, bar, was functionally expressing. Data from DNA hybridization analysis confirmed that the introduced genes (bar and uidA) were integrated into the plant genome and that all lines derived from independent transformation events. Transmission of the introduced genes and the functional expression of bar in T₁ progeny was also confirmed. Germination of T₁ immature embryos in the presence of bialaphos was used as a screen for functional expression of bar; however, leaf painting of T₁ plants proved a more accurate predictor of bar expression in plants. This study suggests that maize Type I callus can be transformed efficiently through microprojectile bombardment and that fertile transgenic plants can be recovered. This system should facilitate the direct introduction of agronomically important genes in to commercial genotypes.     

New vector system for induction of gene expression in dicotyledonous plants

A system of two vectors, pEnLox and pCre, was developed. The pEnLox vector is used to induce insertional mutations, while pCre is used to obtain transgenic plants expressing the Cre recombinase gene. The T-DNA enhancer is flanked by the loxP sites in the pEnLox vector. As an Arabidopsis thaliana insertional mutant obtained by transformation with pEnLox is crossed with another transgenic line carrying the cre gene, the enhancer sequence is eliminated. The vector system makes it possible to induce insertional mutations and to determine whether the mutant phenotype is caused by the loss-of-function insertional mutation or by overespression of nearby genes in response to the enhancer contained in the insert.