Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrids)

Sugarcane (Saccharum spp. hybrids) is an interspecific hybrid with a highly polyploid and frequently aneuploid genome. This C4 grass accounts for nearly 70% of the global sugar production and more recently has become an important biofuel feedstock. Biolistic gene transfer of plasmid DNA is the most frequently used approach for genetic transformation of sugarcane. Minimal expression cassettes lacking vector backbone sequences (MC) have been reported to support simple transgene integration in other species. In this study, we introduced a MC of nptII into embryogenic callus derived from immature leaf whorl cross-sections by biolistic gene transfer. The precipitation equivalents of 12.5, 25 or 50 ng of the nptII MC were delivered per shot to the target tissue with 1.0 μm gold particles. A total of 203 independent putative transgenic plants were regenerated following 80 bombardments and selection on geneticin or paromomycin containing media and 176 transgenic lines were confirmed with PCR. Twenty independent transgenic lines were selected for Southern blot analysis and expression analysis by NPTII ELISA from each of the three treatments. Genomic DNA from transgenic sugarcane plants displayed two to 13 nptII hybridization signals on Southern blots. There was a trend toward reduced transgene integration complexity and reduced transgene expression levels when lower (12.5 ng) MC was used per shot. These results demonstrate that backbone free MCs can be efficiently integrated and expressed in sugarcane.     

Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus

The use of Agrobacterium for gene targeting in plants has been investigated. Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions in the plant genome, were transformed via Agrobacterium with a T-DNA containing a npt-II repair gene. After selection for kanamycin resistance and PCR analysis six recombinants were derived from four of the target lines. The recombination frequencies were similar for the different target lines with one recombinant from approximately 3×10⁵ transformants. Apparently gene targeting is more or less independent of the location of the target construct in the plant genome. Molecular analysis revealed that gene targeting had occurred in five of the six recombinant lines. However precise recombination had occurred in only one line, while in the other four lines restoration of the npt-II gene was accompanied by a deletion of part of the target locus. The sixth recombinant line showed restoration of the npt-II gene of the incoming T-DNA construct which was inserted in the plant genome at a position closely linked to the target locus. The different recombination products favour a model in which recombination is via gene conversion followed by reintegration of the synthesized DNA via homologous or illegitimate recombination rather than a reciprocal exchange of DNA between two cross-overs.     

Transgenic resistance to <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> in <Emphasis Type="Italic">Dendrobium</Emphasis> expressing the viral capsid protein gene

A Taiwan isolate of Cymbidium mosaic virus (CymMV-CS) was isolated from infected Cymbidium sinesis Willd. The cDNA of the capsid protein (CP) gene was synthesized and sequenced. Alignment of this CP gene with other reported CPs revealed homologies of 92–98% at the nucleotide level and 98–99% at the amino acid level. To generate virus-resistant varieties, the CymMV-CS CP gene was transformed into Dendrobium protocorms through particle bombardment. Transformants were selected on medium supplemented with 20 mg/L hygromycin and the presence of the transgene was confirmed by polymerase chain reaction, Southern, Northern and Western blot analyses. Transgenic Dendrobium harboring the CymMV CP gene expressed a very low level of virus accumulation four months post-inoculation with CymMV, as detected by ELISA. The transgenic plants exhibited much milder symptoms than the non-transgenic plants upon challenge with CymMV virionsSequence data reported from this article have been deposited at the GenBank Data Libraries under Accession No. AY429021.     

The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida

The regions of integration of a transferred DNA-fragment from three transgenic Petunia hybrida plants were analysed for their influence on the expression of the foreign DNA. Each of the three transformants, lines 16, 17 and 24, contained a fragment of a plasmid on which two genes were located, an npt-II gene which renders the plants resistant to kanamycin and the A1 gene from Zea mays, a visible marker gene that leads to the production of a brick red anthocyanin pigment in the flowers. Inactivation of both genes in line 16 is associated with integration into a region of highly repetitive DNA, while the integration sites of the other two lines were essentially unique. The integration regions of lines 17 and 24, both of which show expression of the foreign genes at characteristically different intensities, showed a distinct methylation pattern that was stably conserved for these regions in both transgenic and wild-type plants. The characteristic methylation pattern of the two integration regions was also imposed on the border region of the integrated fragments and might thus be responsible for the differences in the intensity of gene expression observed among the two lines.     

Characterization of SMV resistance of soybean produced by genetic transformation of SMV-CP gene in RNAi

Soybean mosaic virus (SMV), a species of the Potyvirus genus in the Potyviridae family, is one of the most typical viral diseases and results in yield and quality loss of cultivated soybean. Due to the depletion of genetic resources for resistance breeding, a trial of genetic transformation to improve disease resistance has been performed by introducing the SMV-CP gene by the RNA interference (RNAi) method via Agrobacterium-mediated transformation. Among 30 transgenic plants produced, 7 lines with enough seeds were infected with SMV and two lines (3 and 4) showed viral resistance to SMV infection. In genomic Southern blot analysis, all the lines tested contained at least one T-DNA insertion. Subsequent investigation confirmed that no viral CP gene expression was detected in two SMV-resistant lines after artificial inoculation of SMV, while non-transgenic control and other transgenic lines expressed substantial amounts of the viral gene. Viral symptoms affected seed morphology, and clean seeds were harvested from the resistant lines. Also, strong viral gene expression was detected from the seeds of susceptible lines. In further generations, the same phenotypic appearance was maintained among non-transgenic and transgenic plants. Finally, the presence of helper component-proteinase (HC-Pro), known as a suppressor of gene silencing apparatus, was checked among transgenic lines. No expression of HC-Pro in resistant lines indicated that the viral CP-RNAi transformation into soybean somehow created a functional gene silencing system and resulted in a viral-resistant phenotype.     

Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis

A protocol is described that supports the production of transgenic sugarcane plants ready for transfer to soil within 3 mo from culture initiation. Biolistic gene transfer into cross-sections of immature leaf whorl explants followed by direct somatic embryogenesis resulted in the stable genetic transformation of the commercially important sugarcane cultivar CP 88-1762. Accelerating the production of transgenic sugarcane plants not only saves time and effort but will likely also minimize somaclonal variation. Southern blot analysis revealed simple transgene integration patterns ranging from one to five hybridization products. NPTII-ELISA confirmed that most of the transgenic plants expressed the transgene stably in vegetative progeny. Using a minimal, linear expression cassette (MC) without vector backbone sequences for the biolistic gene transfer and reducing the amount of MC to 10 ng per shot may have led to simple transgene integration and stable transgene expression. Therefore, this protocol has great potential for the generation of commercial transgenic sugarcane events.     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.     

Characterization of T-DNA insertions in transgenic grapevines obtained by Agrobacterium-mediated transformation

T-DNA integration patterns in 49 transgenic grapevines produced via Agrobacterium-mediated transformation were analyzed. Inverse PCR (iPCR) was performed to identify T-DNA/plant junctions. Sequence comparison revealed several deletions in the T-DNA right border (RB) and left border (LB), and filler DNA and duplications or deletions of grapevine DNA at the T-DNA insertion loci. In 20 T-DNA/grapevine genome junctions microsimilarities were found associated with the joining points and in all grapevine lines microsimilarities were present near the breaking points along the 30 bases of T-DNA adjacent to the two borders. Analysis of target site preferences of T-DNA insertions indicated a non-random distribution of the T-DNA, with a bias toward the intron regions of the grapevine genes. Compositional analysis of grapevine DNA around the T-DNA insertion sites revealed an inverse relationship between the CG and AT-skews and AT rich sequences present at 300–500 bp upstream the insertion points, near the RB of the T-DNA. PCR assays showed that vector backbone sequences were integrated in 28.6% of the transgenic plants analyzed and multiple T-DNAs frequently integrated at the same position in the plant genome, resulting in the formation of tandem and inverted repeats.     

Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.     

Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.     

Effect of transgenesis on reproductive traits of rabbit males

The influence of foreign transgene integration on the reproductive capabilities of rabbit males is not known. Therefore, we analyzed their ejaculate characteristics, reproductive capabilities, occurrence of pathological spermatozoa and histological structure of the testis. We have generated transgenic rabbits by microinjection of WAP-hFVIII gene into pronucleus of fertilized egg. We observed that the libido, volume and pH value of the ejaculate did not differ significantly between transgenic and non-transgenic male lines. The motility, concentration, osmolarity, thermoresistant test of spermatozoa (at 1 or 6 h) and the percentage of alive spermatozoa were significantly different (p < 0.001) among transgenic and non-transgenic males. No significant differences were found between transgenic and non-transgenic male lines in the occurrence of pathological spermatozoa and histology of the testis. The ability of spermatozoa from transgenic and non-transgenic males to fertilize eggs was ranged within 96 and 100%; while the yield of transgenic embryos ranged from 43 to 57%. Our results show that mammary gland specific over-expression mWAP-hFVIII gene construct does not affect reproductive traits of transgenic rabbit males.     

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene

Summary Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and -glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.     

MicroTom—a high-throughput model transformation system for functional genomics

We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%, in industrial production scale transformation experiments. For CP4, with glyphosate selection, the average transformation frequency was 57%. Stable transformation frequency was positively correlated with transient expression (R=0.85), and variable with the genes of interest. DNA integration and germline transformation were confirmed by biological assay, Southern Blot analysis, and R₁ phenotype segregation. Transgene expression was observed in leaf, root, stem, flower, and fruit tissues of the transgenic plants. Ninety-five percent of transgenic events coexpressed two introduced genes based on β-glucuronidase (GUS) and neonmycin phosphotransferase II (NPTII) expression. Seventy-five percent of transgenic events contained one to two copies of the introduced uidA (GUS) gene based on Southern analysis. Transgenic plants from the cotyledon explants to the transgenic plants transferred to soil were produced within about 2–3 months depending on the genes of interest. The utility of this MicroTom model transformation system for functional genomic studies, such as identification of genes related to important agricultural traits and gene function, is discussed.