Genetic transformation of the oilseed crop <Emphasis Type="Italic">Crambe abyssinica</Emphasis>

Engineering oilseed crops for industrial purposes requires a suitable crop that does not outcross to any food oilseed crop, thus eliminating problems of gene flow. Crambe abyssinica is such a dedicated crop as it does not hybridize with any existing food oilseed crops. However, lack of regeneration and transformation protocols has limited the use of C. abyssinica in genetic manipulation studies. In this study, efficient regeneration and transformation protocols for Crambe have been developed. Hypocotyls of C. abyssinica cv. Galactica were incubated on a Murashige and Skoog medium supplemented with various plant growth regulators (PGRs). Among the different PGR combinations tested, 10 μM thidiazuron and 2.7 μM α-naphthaleneacetic acid promoted highest frequency of regeneration, up to 60%. Among six Agrobacterium stains evaluated, each harbouring the cloning vector containing the neomycin phosphotransferase (nptII) and β-glucuronidase (gus) genes. EHA101 and AGL-1 yielded the highest transformation frequencies of 1.3 and 2.1%, respectively. Putative transgenic lines were recovered, and confirmed as transgenic by Southern blot analysis. Subsequently, Agrobacterium-mediated transformation of hypocotyls of cv. Galactica with constructs harbouring the wax synthase and fatty acid reductase genes have also successfully recovered confirmed transgenic plants carrying these transgenes.     

Strategies for developing marker-free transgenic plants

The development of marker-free transgenic plants has responded to public concerns over the safety of biotechnology crops. It seems that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning the public acceptability of transgenic crop plants. Selectable marker genes are co-introduced with genes of interest to identify those cells that have integrated the DNA into their genome. Despite the large number of different selection systems, marker genes that confer resistance to the antibiotics, hygromycin (hpt) and kanamycin (nptII) or herbicide phosphinothricin (bar), have been used in most transgenic research and crop development techniques. The techniques that remove marker gene are under development and will eventually facilitate more precise and subtle engineering of the plant genome, with widespread applications in both fundamental research and biotechnology. In addition to allaying public concerns, the absence of resistance genes in transgenic plants could reduce the costs of developing biotechnology crops and lessen the need for time-consuming safety evaluations, thereby speeding up the commercial production of biotechnology crops. Many research results and various techniques have been developed to produce marker-free transgenic plants. This review describes the strategies for eliminating selectable marker genes to generate marker-free transgenic plants, focusing on the three significant marker-free technologies, co-transformation, site-specific recombinase-mediated excision, and non-selected transformation.     

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 × 10⁶-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 × 10³ genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR). Received: 20 May 1997 / Accepted: 17 October 1997     

Reconsideration of pollen dispersal data from field trials of transgenic potatoes

During the initial field evaluation of transgenic plants, it is usual to isolate them genetically from other plants of the same species. Several field experiments on potatoes, using transgenes as markers, have shown that transgene dispersal by pollen to other potato plants is limited and very unlikely at distances over 10 m. In a recent study in Sweden, a frequency of transgene-containing progeny of over 30% is reported from non-transgenic potato plants grown at distances of 10–1000 m from transgenic plants containing nptII and gus marker genes. Data from the Swedish study is discussed along with other relevant observations, and it is concluded that the high frequency of gene dispersal in that study results from a high frequency of false positives during PCR analysis of the nptII gene. From the data available in potato, it is concluded that a distance of 20 m is generally adequate for the initial field evaluation of transgenic potatoes containing novel gene constructs.     

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6?×?10⁶-fold excess of plant DNA. Only 18 ng of potato DNA (2.5?×?10³ genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR).     

Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.     

Environmental fate and behaviour of antibiotic resistance genes and small interference RNAs released from genetically modified crops

Rising global populations have amplified food scarcity across the world and ushered in the development of genetically modified (GM) crops to overcome these challenges. Cultivation of major crops such as corn and soy has favoured GM crops over conventional varieties to meet crop production and resilience needs. Modern GM crops containing small interference RNA molecules and antibiotic resistance genes have become increasingly common in the United States. However, the use of these crops remains controversial due to the uncertainty regarding the unintended release of its genetic material into the environment and possible downstream effects on human and environmental health. DNA or RNA transgenes may be exuded from crop tissues during cultivation or released during plant decomposition and adsorbed by soil. This can contribute to the persistence and bioavailability in soil or water environment and possible uptake by soil microbial communities and further passing of this information to neighbouring bacteria, disrupting microbial ecosystem services such as nutrient cycling and soil fertility. In this review, transgene mechanisms of action, uses in crops, and knowledge regarding their environmental fate and impact to microbes are evaluated. This aims to encapsulate the current knowledge and promote further research regarding unintended effects transgenes may cause.     

Unsuccessful search for DNA transfer from transgenic plants to bacteria in the intestine of the tobacco horn worm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

DNA transfer from transgenic plants to native intestinal bacteria and introduced Acinetobacter BD413 was assessed in the gut of the tobacco horn worm (Manduca sexta). The marker was kanamycin resistance gene (nptII), and tobacco carrying the nptII gene in the chloroplasts served as the donor. We detected neither whole gene transfer to native bacteria, nor transfer of fragments of nptII to Acinetobacter, using a marker exchange assay. This negative result was attributed to a heat-labile activity that degraded DNA in the feces, probably DNAase. Nevertheless, a few intact leaf cells survived transit through the gut, and DNA extracted from feces did transform Acinetobacter, albeit at lower frequencies than DNA extracted from leaves.     

Abundances of Tetracycline,Sulphonamide and Beta-Lactam Antibiotic Resistance Genes in Conventional Wastewater Treatment Plants (WWTPs) with Different Waste Load

Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs), an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs) were quantified using qPCR in three WWTPs of decreasing capacity located in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2), tetracycline resistance genes (tetM and tetC), and resistance genes for extended spectrum beta-lactams (bla_oxa-58, bla_shv-34, and bla_ctx-m-32). To avoid inconsistencies among qPCR assays we normalised the ARG abundances with 16S rRNA gene abundances while assessing if the respective genes increased or decreased during treatment. ARGs were detected in most samples; sul1, sul2, and tetM were detected in all samples. Statistically significant differences (adjusted p<0.01) between the inflow and effluent were detected in only four cases. Effluent values for bla_oxa-58 and tetC decreased in the two larger plants while tetM decreased in the medium-sized plant. Only bla_shv-34 increased in the effluent from the medium-sized plant. In all other cases the purification process caused no significant change in the relative abundance of resistance genes, while the raw abundances fell by several orders of magnitude. Standard water quality variables (biological oxygen demand, total phosphorus and nitrogen, etc.) were weakly related or unrelated to the relative abundance of resistance genes. Based on our results we conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs.     

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.     

Nucleotide substitutions in <Emphasis Type="Italic">rolC</Emphasis> and <Emphasis Type="Italic">nptII</Emphasis> gene sequences during long-term cultivation of <Emphasis Type="Italic">Panax ginseng</Emphasis> cell cultures

It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P. ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1–4 nucleotide substitutions were found per sequence in the 540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted from P. ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 ± 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased (1.37 ± 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P. ginseng actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred genes in transgenic plant cell cultures.     

Bioactive beads-mediated transformation of rice with large DNA fragments containing Aegilops tauschii genes

Transformation with large DNA molecules enables multiple genes to be introduced into plants simultaneously to produce transgenic plants with complex phenotypes. In this study, a large DNA fragment (ca. 100 kb) containing a set of Aegilops tauschii hardness genes was introduced into rice plants using a novel transformation method, called bioactive beads-mediated transformation. Nine transgenic rice plants were obtained and the presence of transgenes in the rice genome was confirmed by PCR and FISH analyses. The results suggested that multiple transgenes were successfully integrated in all transgenic plants. The expression of one of the transgenes, puroindoline b, was confirmed at the mRNA and protein levels in the T₂ generation. Our study clearly demonstrates that the bioactive bead method is capable of producing transgenic rice plants carrying large DNA fragments. This method will facilitate the production of useful transgenic plants by introducing multiple genes simultaneously.     

Genomic DNA can be used with cationic methods for highly efficient transformation of maize protoplasts

Summary Efficient delivery of genomic DNA fragments to maize protoplasts was obtained by new methods using the polycation Polybrene or Lipofectin cationic liposomes. Stable kanamycin-resistant secondary transformants were recovered after transfection with genomic DNA from a maize cell line that had previously been tagged with the bacterial gene neomycin phosphotransferase (nptII) in a first-round transformation. The frequency of secondary transformants with nptII-homologous DNA sequences was 3% or 6% of all randomly picked microcalli after Polybrene or Lipofectin-mediated transfection, respectively. Transformation with genomic DNA by these methods may allow easy transfer of uncloned genes encoding desirable characteristics to crop species that can be regenerated from protoplasts.     

Evaluation of possible horizontal gene transfer from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413

 The use of genetically engineered crop plants has raised concerns about the transfer of their engineered DNA to indigenous microbes in soil. We have evaluated possible horizontal gene transfer from transgenic plants by natural transformation to the soil bacterium Acinetobacter calcoaceticus BD413. The transformation frequencies with DNA from two sources of transgenic plant DNA and different forms of plasmid DNA with an inserted kanamycin resistance gene, nptII, were measured. Clear effects of homology were seen on transformation frequencies, and no transformants were ever detected after using transgenic plant DNA. This implied a transformation frequency of less than 10^-13 (transformants per recipient) under optimised conditions, which is expected to drop even further to a minimum of 10^-16 due to soil conditions and a lowered concentration of DNA available to cells. Previous studies have shown that chromosomal DNA released to soil is only available to A. calcoaceticus for limited period of time and that A. calcoaceticus does not maintain detectable competence in soil. Taken together, these results suggest that A. calcoaceticus does not take up non-homologous plant DNA at appreciable frequencies under natural conditions. Received: 1 November 1996 / Accepted: 18 April 1997     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.     

Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia

Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.     

Genetic transformation of cork oak (Quercus suber L.) for herbicide resistance

The bar gene was introduced into the cork oak genome. Cork oak embryogenic masses were transformed using the Agrobacterium strain AGL1 which carried the plasmid pBINUbiBar. This vector harbours the genes, nptII and bar, the latter under control of the maize ubiquitin promoter. The transgenic embryogenic lines were cryopreserved. Varying activities of phosphinothricin acetyl transferase were detected among the lines, which carried 1–4 copies of the insert. Molecular and biochemical assays confirmed the stability and expression of the transgenes 3 months after thawing the cultures. These results demonstrate genetic engineering of herbicide tolerance in Quercus spp. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Rubén álvarez, Ricardo J. Ordás are contributed equally.     

Marker-free transgenic corn plant production through co-bombardment

The use of particle gun for the production of marker-free plants is scant in published literature. Perhaps this is a reflection of the widely held notion that the events generated through bombardment tend to have multiple copies of transgenes, usually integrated at a single locus, features which precludes segregating away the selectable marker gene. However, our previous studies have shown that single-copy integrants are obtained at a high frequency if limited quantity of DNA is used for bombardment. Also, the concatemerized insertion of transgenes has been demonstrated to be greatly reduced if “cassette DNA” is employed in place of whole plasmid DNA for bombardment. Based on the above findings, in the present study the feasibility of co-bombardment was evaluated for the production of marker-free plants in corn, employing a combination of limited quantity DNA and cassette DNA approaches for bombardment. Transgenic events were generated after co-bombardment of a selectable marker cassette containing the nptII gene (2.5 ng per shot) and a GUS gene cassette (15 ng per shot). Among these events single-copy integrants for nptII gene occurred at an average frequency of 68% within which the co-expression frequency of GUS and nptII genes ranged from 41% to 80%. Marker-free corn plants could be identified from the progeny of 28 out of the 103 R0 co-expressing events screened. The results demonstrate that by using cassette DNA and low quantities of DNA for bombardment, marker-free plants are produced at efficiencies comparable to that of Agrobacterium-based co-transformation methods.