Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome

The bacteriophage P1 Cre—lox site-specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant lox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild-type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre—lox site-specific recombination system is useful for the single-copy integration of DNA into a chromosomal lox site.     

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.     

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.     

Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.     

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     

Cre-mediated seed-specific transgene excision in tobacco

Here we report the production of marker-free transgenic plants expressing phenolic compounds with high pharmacological value. Our strategy consisted in simultaneous delivery of lox-target and cre-containing constructs into the plant genome by cotransformation. In the Cre-vector, the cre recombinase gene was controlled by a seed-specific napin promoter. In the lox-target construct the selectable bar gene was placed between two lox sites in direct orientation, while a napin promoter driven vstI gene was inserted outside of the lox sites. Upon seed-specific cre induction the bar expression cassette was excised from the tobacco genome. Genetic and molecular analysis of T1 progeny plants indicated DNA excision in all 10 transgenic lines tested. RP-HPLC analysis demonstrated that the expression of the vstI gene resulted in accumulation of trans-resveratrol and its glycosylated derivative piceid in seeds of all marker free lines. These findings indicate that the seed-specific marker gene excision did not interfere with the expression of the gene of interest. Our data demonstrated the feasi of a developmentally controlled cre gene to mediate site-specific excision in tobacco very efficiently.     

Inheritance of microspore embryogenic ability in Brassica crops

Inheritance of microspore embryogenic ability in oilseed rape (Brassica napus L.) and Chinese cabbage (Brassica campestris L. ssp. pekinensis) was examined by 4 × 4 diallel crosses using cultivars showing a different response. In both species, embryo yields of most F₁ hybrids were similar to, or over, the high responsive parent and some F₁s showed intermediate embryo yields between their parents. Diallel analysis showed that both additive and dominant effects were significant at the 1% level for the genetic control of microspore embryogenic ability in both species. Dominant genes had positive effects on microspore embryogenesis. In oilseed rape, the additive effects were important, while in Chinese cabbage the dominant effects were largely contributed. The broad- and narrow-sense heritabilities were 0.972 and 0.811 in oilseed rape, and 0.959 and 0.659 in Chinese cabbage, respectively. From the results of the segregation of embryo yields in the F₂ population of ’Lisandra’×’Kamikita’, it is considered that the microspore embryogenic ability is controlled by two loci with additive effects in oilseed rape. Received: 6 September 2000 / Accepted: 24 November 2000     

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.     

A Self-Excising Cre Recombinase Allows Efficient Recombination of Multiple Ectopic Heterospecific Lox Sites in Transgenic Tobacco

To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox sites, either lox511 or lox2272 or both, in trans. The plant intron-containing cre gene, cre ^INT , was configured in such a way that self-excision generated an intact hygromycin resistance selectable marker gene. In this combination, all selected transformants showed highly efficient excision. Plants obtained showed no indication of any chimerism, indicating a cell autonomous nature of the hygromycin selection during transformation and regeneration. The highly efficient concomitant removal of wildtype and heterospecific lox site-flanked DNA demonstrated that upon retransformation with the self-excising cre ^INT , sufficient amounts of Cre enzyme were produced prior to its removal. Plants obtained with cre ^INT showed much less frequently the Cre-associated phenomenon of reduced fertility than plants obtained with a continuous presence of Cre recombinase. The cre ^INT system has therefore advantages over systems with a continuously present Cre. The cre ^INT system was successfully used for removal of two chromatin boundary elements from transgene cassettes in tobacco. Analysis of plants with and without boundary elements on the same chromosomal location will contribute to a better evaluation of the role of such elements in the regulation of transgene expression in 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.     

Evaluation of four grasses for use in phytoremediation of Cs-contaminated arid land soil

Although residue management seems a key factor in residue-mediated weed suppression, very few studies have systematically compared the influence of different residue management strategies on the establishment of crop and weed species. We evaluated the effect of several methods of pre-treatment and placement of winter rye (Secale cereale L.) and winter oilseed rape (Brassica napus L.) residue on seedling emergence under field conditions. For both species two cultivars, differing in allelochemical content, were used. Residues incorporated in the upper soil layer exerted a large inhibitory effect on the establishment of the relatively early emerging lettuce (Lactuca sativa L.) and spinach (Spinacia oleracea L.) seedlings, whereas the inhibitory effect on the slightly later emerging Stellaria media L. seedlings was variable, and often a stimulatory effect on the very late emerging Chenopodium album L. seedlings was observed. Differences between cover crop cultivars were minor. For winter oilseed rape residue, pre-treatment strongly affected the time-course of residue-mediated effects. Finely ground residues were only inhibitory to seedling establishment during the first two to three weeks, whereas cut residues became inhibitory after this period. For winter rye, residue placement was most important. Residue incorporation gave variable results, whereas placement of winter rye residue on top of the soil inhibited the emergence of all receptor species. In conclusion, the optimal residue management strategy for weed suppression depends both on the cover crop species used and the target weed species.     

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Heat-inducible Cre-<Emphasis Type="Italic">lox</Emphasis> system for marker excision in transgenic rice

The present study assessed the efficacy of a heat-inducible cre gene for conditional removal of the marker gene from a rice genome via Cre-lox recombination. A cre gene controlled by the soybean heat-shock promoter was introduced into the rice genome along with the recombination target (lox) construct. Cre-mediated recombination was expected to remove the marker gene and activate the promoter-less GUS gene. Six transgenic lines displayed well-regulated heat-inducible Cre activity in the callus. However, only one line that contained a single copy of the cre gene maintained this property in the regenerated plants and their progeny. Marker-free progeny were obtained from the plant that was heat-treated at the seedling stage, indicating the inheritance of the recombination ‘footprint’. The presence of the ‘footprint’ was verified by polymerase chain reaction and Southern analysis. Therefore, the cre gene controlled by the soybean heat-shock promoter is an effective tool for conditional removal of the marker gene in rice.     

Directed excision of a transgene from the plant genome

Summary The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, -glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALS^r), were integrated into the genome of tobacco and Arabidopsis. The ALS^r gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALS^T gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The -glucuronidase gene remained active. The excision efficiency varied in F₁ progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F₁ progeny were chimeric and some F₂ progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.     

Molecular Cytogenetic Localization and Characterization of 5S and 25S rDNA Loci for Chromosome Identification in Oilseed Rape (Brassica napus L.)

Chromosome identification in oilseed rape (Brassica napus L.)is extremely difficult using conventional cytogenetic techniquesbecause amphidiploid Brassica species possess numerous verysmall chromosomes with few cytogenetic landmarks. In combinationwith methods for improved chromosome preparations, we used asimplified fluorescencein situ hybridization (FISH) techniqueto localize simultaneously the gene families coding for 5S and25S rDNA in B. napus. The resulting hybridization patterns enabledten of the 19 oilseed rape chromosome pairs to be unequivocallyidentified. Copyright 2000 Annals of Botany Company Brassica napus, oilseed rape, rDNA, molecular cytogenetics, FISH, chromosome identification     

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. ‘Grande Naine’ (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.     

Exploitation of host plant preferences in pest management strategies for oilseed rape (Brassica napus)

New control strategies for insect pests of arable agriculture are needed to reduce current dependence on synthetic insecticides, the use of which is unsustainable. We investigated the potential of a simple control strategy to protect spring‐sown oilseed rape, Brassica napus L. (Brassicaceae), from two major inflorescence pests: the pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), and the seed weevil, Ceutorhynchus assimilis (Paykull) (Coleoptera: Curculionidae), through exploitation of their host plant preferences. The strategy comprised, for the main crop, Starlight [an oilseed rape cultivar with relatively low proportions of alkenyl glucosinolates in the leaves (thereby releasing lower levels of attractive isothiocyanates than conventional cultivars)] and turnip rape, Brassica rapa (L.) (Brassicaceae), as a trap crop. We tested the system in laboratory, polytunnel semifield arena, and field experiments. The odours of Starlight were less attractive in olfactometer tests to both pests than those from a conventional cultivar, Canyon, and the plants were less heavily colonized in both polytunnel and field experiments. Turnip rape showed good potential as a trap crop for oilseed rape pests, particularly the pollen beetle as its odour was more attractive to both pests than that of oilseed rape. Polytunnel and field experiments showed the importance of relative growth stage in the system. As turnip rape flowers earlier than oilseed rape, beetles would be maintained on turnip rape past the damage‐susceptible growth stage of oilseed rape. The development of a pest control regime based on this strategy is discussed.     

Deletion of IL-4 Receptor Alpha on Dendritic Cells Renders BALB/c Mice Hypersusceptible to Leishmania major Infection

In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11c^creIL-4Rα^-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11c^creIL-4Rα^-/lox mice. Following infection with L. major, CD11c^creIL-4Rα^-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11c^creIL-4Rα^-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11c^creIL-4Rα^-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions.     

Temporal changes in climatic variables and their impact on crop yields in southwestern China

Knowledge of variability in climatic variables changes and its impact on crop yields is important for farmers and policy makers, especially in southwestern China where rainfed agriculture is dominant. In the current study, six climatic parameters (mean temperature, rainfall, relative humidity, sunshine hours, temperature difference, and rainy days) and aggregated yields of three main crops (rice: Oryza sativa L., oilseed rape: Brassica napus L., and tobacco: Nicotiana tabacum L.) during 1985–2010 were collected and analyzed for Chongqing—a large agricultural municipality of China. Climatic variables changes were detected by Mann-Kendall test. Increased mean temperature and temperature difference and decreased relative humidity were found in annual and oilseed rape growth time series (P?<?0.05). Increased sunshine hours were observed during the oilseed rape growth period (P?<?0.05). Rainy days decreased slightly in annual and oilseed rape growth time series (P?<?0.10). Correlation analysis showed that yields of all three crops could benefit from changes in climatic variables in this region. Yield of rice increased with rainfall (P?<?0.10). Yield of oilseed rape increased with mean temperature and temperature difference but decreased with relative humidity (P?<?0.01). Tobacco yield increased with mean temperature (P?<?0.05). Path analysis provided additional information about the importance and contribution paths of climatic variables to crop yields. Temperature difference and sunshine hours had higher direct and indirect effects via other climatic variables on yields of rice and tobacco. Mean temperature, relative humidity, rainy days, and temperature difference had higher direct and indirect effects via others on yield of oilseed rape.