Radiation as a tool to remove selective marker genes from transgenic soybean plants

The present study evaluated the use of γ-radiation to physically remove selective marker genes previously introduced into the soybean genome. Homozygous seeds from a transgenic soybean line carrying the gus and ahas transgenes were irradiated with γ-rays. Six plants presenting a deleted gus gene were analyzed by Southern blot to confirm removal of both ahas and gus genes. Line 1A presented an absence of the gus gene cassette and presence of the ahas gene cassette.     

Efficient selection of transgenic mouse embryos using EGFP as a marker gene

We have established a reliable method that uses the EGFP (Enhanced Green Fluorescent Protein) gene as a marker for selecting transgenic embryos from preimplantation embryos. Embryos that were subjected to the pronuclear microinjection of the CMV/β‐actin/EGFP fusion gene were cultured in vitro until they developed into the morulae‐ or blastocyst‐stage. The expression of EGFP was easily observed by a fluorescent microscopy. There appeared to be no damage to the in vivo developmental ability of the embryos in response to the EGFP excitation light, which utilized an IB filter for a period of 30 min. Modified PCR analysis using Dpn I and Bal 31 digestion of the embryonic DNA showed that all of the embryos expressing EGFP in all their cells were transgenic, while more than half with mosaic expression of EGFP were not transgenic. Approximately 77% of pups born from the embryos that uniformly expressed the EGFP gene were transgenic, while 21.4% of pups from the embryos with mosaic expression were transgenics. The results showed that the use of EGFP as a marker is very useful and reliable for selecting transgenic embryos, and that it is important to transfer the embryos expressing EGFP in all their cells to obtain truly transgenic animals. Mol. Reprod. Dev. 54:43–48, 1999. © 1999 Wiley‐Liss, Inc.     

Genetic transformation of Cymbidium orchid by particle bombardment

 A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse. Received: 20 July 1998 / Revision received: 2 December 1998 / Accepted: 17 December 1998     

Chlorsulfuron resistant transgenic tobacco as a tool for broomrape control

Broomrape (Orobanche ramosa L.) is the most important parasitic plant that infests tobacco (Nicotiana tabacum L.). Chemical treatment of the soil is not effective and crop rotation is not acceptable to solve this problem because of the long viability period of Orobanche seeds in the soil. Application of systemic herbicides in the field with herbicide resistant tobacco could be a successful tool for broomrape control. Several tobacco cultivars were transformed with a mutant ahas3R gene for resistance to the herbicide chlorsulfuron (Glean^®, DuPont). Transformed plants were selfed and the segregation of resistance was followed in the next generation. The efficiency of the herbicide was demonstrated in greenhouse and field trials. An Orobanche/tobacco growth system was used in order to prove the lethal effect of the herbicide to the attached broomrape plants.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

Kanamycin resistance of germinating pollen of transgenic plants

The influence of kanamycin on the percentage of pollen germination and on tube growth of pollen from non-transformed and transformed plants of various species containing a chimaeric kanamycin resistance gene (NPTII) was investigated. Pollen grains isolated from kanamycin resistant plants expressed resistance when germinating in vitro, whereas kanamycin impaired tube growth of pollen from non-transformed plants. Pollen grains from transgenic plants were less sensitive and produced significantly longer tubes. mRNAs of the chimaeric gene are probably presynthesized concurrently with the other mRNAs during microsporogenesis, and kanamycin resistance is expressed by mRNA translation during pollen tube elongation. Received: 24 August 1999 / Revision accepted: 20 October 1999     

Agrobacterium-mediated transformation and plant regeneration of Brassica oleracea var. capitata

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of four commercial cultivars of Brassica oleracea var. capitata is described. A strain of A. tumefaciens LBA4404 with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette were used for co-cultivation. Preliminary selection of regenerated transgenic plants was performed on kanamycin-containing medium. The frequency of transgenic plants was calculated on the basis of GUS (β-glucuronidase) activity detected by the histochemical X-gluc test. Tissue-specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. The transformation rates of the commercial cultivars of B. oleracea was higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and multiple loci in the genome. All transgenic plants grew normally after a brief vernalization period and showed stable inheritance of the marker gene. The present study demonstrates that morphologically normal, fertile transgenic plants of B. oleracea can be obtained. Received: 24 August 1999 / Revision received: 23 November 1999 / Accepted: 3 December     

Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus

 The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performing β-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV-CP gene varied from one to six among the transgenic lines. Half of them showed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or integration pattern. Received: 7 April 1999 / Revision received: 17 June 1999 · Accepted: 24 June 1999     

Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants

A genomic clone encoding the potato homolog of the yeast ubiquitin-ribosomal protein fusion gene ubi3 was isolated and characterized. Chimeric genes containing the ubi3 promoter (920 bp of 5 to the ubiquitin start codon) were constructed in which the reporter gene -glucuronidase (GUS) was either fused directly to the promoter, or introduced as a translational fusion to the ubiquitin-coding region. After introduction into the potato by Agrobacterium-mediated transformation, GUS activities were measured in leaves and in tubers of transgenic clones. GUS activity was 5- to 10-fold higher in clones expressing the ubiquitin-GUS translational fusion than in clones containing GUS fused directly to the ubi3 promoter. For both types of constructs, GUS activity was highest in meristematic leaves and declined during leaf expansion, then rose again to near the meristematic levels during senescence. GUS activity in tubers was similar to that in young leaves. In contrast to the native ubi3 genes, the chimeric ubi3-GUS transgenes were not activated in the tuber by wounding.     

Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the “Class II” hemoglobins. Received: 28 June 1999 / Accepted: 25 November 1999     

The promoter of the gene Itr1 from barley confers a different tissue specificity in transgenic tobacco

Tissue-specific expression of the gene coding for trypsin inhibitor BTI-CMe in barley (Itr1) occurs during the first half of endosperm development. In transgenic tobacco, theItr1 promoter drives expression of the β-glucuronidase reporter gene not only in developing endosperm but also in embryo, cotyledons and the meristematic intercotyledonary zone of germinating seedlings. A promoter fragment extending 343 bp upstream of the translation initiation ATG codon was sufficient for full transgene expression, whereas, the proximal 83 bp segment of the promoter was inactive. Possible reasons for the differences in expression patterns are discussed. These authors have contributed equally to this work     

Expression of a chimeric nitrate reductase gene in transgenic lettuce reduces nitrate in leaves

 Transgenic plants of four glasshouse-grown lettuce cultivars ('Cortina', 'Evola', 'Flora' and 'Luxor') were obtained by co-cultivating excised cotyledons with Agrobacterium tumefaciens. The Agrobacterium strain LBA4404 contained the binary vector pBCSL16, which carried a nitrate reductase (nia) cDNA linked to CaMV promoter and terminator sequences, and the neomycin phosphotransferase II (nptII) gene. Transformed shoots were selected by their ability to root on medium containing kanamycin sulphate, by a positive NPTII assay and by PCR analysis. The presence of the nia cDNA in transgenic lettuce was confirmed by nitrate reductase (NR) enzymatic assay, a reduction in the nitrate content of leaves and by Southern hybridisation. PCR analysis of cDNA fragments from transgenic plants confirmed that both nia and nptII genes were expressed in first seed-generation (T1) lettuce plants. The commercial importance of reduced nitrate concentrations in lettuce is discussed. Received: 7 January 1998 / Revision received: 24 February 1998 / Accepted: 22 March 1999     

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme

 NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C₄ species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C₄ subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts. Received: 27 January 1999 / Accepted: 20 January 2000     

A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene

The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones. Received: 22 June 1999 / Accepted: 1 September 1999     

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).     

Vegetative and generative dispersal capacity of field released transgenic aspen trees

Transfer of genes by pollen or wind-dispersed seed is considered a main potential risk when field release experiments with transgenic trees are initiated. In Germany, the first release experiment with genetically transformed trees was initiated in 1996. To ensure that the transgenic trees remained in the vegetative phase, the duration of the experiment was limited to 5 years. In total, 457 1-year-old trees including eight transgenic aspen lines carrying either the 35S- rolC or the rbcS- rolC gene construct, and three control clones were transferred to the field. In 1998 and 2000, 12 plants of transgenic lines all carrying the 35S- rolC gene construct formed female flower buds. Furthermore, one young aspen plant identified as a root sucker was observed in 1999 followed by an increasing number of root suckers derived from transgenic and non-transgenic trees in 2000 and 2001. In 2001, the last year of the field trial, 15 root suckers were detected outside the field. In total, 234 root suckers were harvested in 2000 and 2001 and analysed for their transgenic status. More than half of the roots suckers investigated showed the presence of the rbcS- rolC gene construct. We concluded that in addition to the widely accepted generative propagation, vegetative dispersal capacity of transgenic perennial plants is also important and must be included in risk assessment studies.     

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.     

Effective selection system for generating marker-free transgenic plants independent of sexual crossing

 In a previous report, a novel selection protocol termed "the MAT-vector system" for generating marker-free transgenic plants (MFTPs) was presented. The first stage of the system is visual selection of morphologically abnormal transgenic shoots, ipt-shooty, that have lost apical dominance and rooting ability. The second stage involves elimination of the ipt gene and the appearance of MFTPs free of ipt gene influence. The present report describes a practical MAT-vector in which removal of the ipt gene is efficiently mediated by the site-specific recombination system R/RS from Zygosaccharomyces rouxii, in place of the maize transposable element Ac, used previously. This improved MAT-vector produced MFTPs from 39% of moderate ipt-shooty and 70% of extreme ipt-shooty lines. These results are superior to the previous MAT-vector which produced MFTPs from only 5% of ipt-shooty lines. The present novel system also induced direct development of MFTPs from adventitious buds without production of ipt-shooty intermediates. The presence of β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) genes of interest, and the absence of the ipt gene were verified by a GUS histochemical assay, NPTII assay, and molecular analysis. Received: 19 June 1998 / Revision received: 4 December 1998 / Accepted: 18 December 1998     

A protocol for consistent, large-scale production of fertile transgenic rice plants

A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment of embryogenic tissues (Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines carrying the hygromycin phosphotransferase (hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates, when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure, transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0 plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies. Received: 12 March 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998