Agrobacterium-Mediated Transformation of Creeping Bentgrass Using GFP as a Reporter Gene (original) (raw)
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In vivo performance of a dual genetic marker,manA-gfp, in transgenic bentgrass
Genome, 2005
A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated b...
Plant Cell Reports, 1993
Transgenic creeping bentgrass (Agrostis palustris Huds., cv. Pencross; Poaceae) plants have been obtained by microprojectile bombardment of and regeneration from embryogenic calli with a vector designed to deliver the β-glucuronidase (GUS) gene under the control of rice actin 1 5' regulatory sequences. Southern analysis of polymerase chain reaction (PCR)-amplified and restriction-digested genomic DNA of four transgenic plants regenerated from these cultures showed the unscrambled integration of the gus fragment. Northern blot analysis confirmed the expression of gus mRNA in one of the transgenic plants. Western blot analysis revealed a high level of accumulation of gus protein. Histochemical assays showed enzymatic activity of β-glucuronidase in all parts of the transgenic turfgrass plant. The order of gus expression level in different tissues of the transgenic plant is as follows: stem node > first young leaf > root tip > second / third / fourth young leaf > stem internode > root hair-zone.
Plant Cell Reports, 2005
A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment cocultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the β-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD 600 ∼ =0.6 and diluted to a cell density of 10 9 cells ml −1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 µg ml −1 ). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gusintron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l −1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T 1 seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.
Agrobacterium-mediated transformation of plants: Basic principles and influencing factors
African Journal of …, 2011
Transformation is an important topic in plant biology and transgenic plants have become a major focus in plant research and breeding programs. Agrobacterium-mediated transformation as a practical and common method for introducing specific DNA fragments into plant genomes is well established and the number of transgenic plants produced using this method is increasing. Despite the popularity of the method, low efficiency of transformation is a major challenge for scientists. Modification of different genetic and environmental aspects of transformation method may lead to better understanding of the system and result in high efficiency transformation. In this review, we deal with recent genetic findings as well as different environmental factors which potentially influence Agrobacterium-mediated transformation.
In Vitro Cellular & Developmental Biology - Plant, 2009
Until recently, information about the effects of transforming plants with the rolA gene of Agrobacterium rhizogenes has been restricted mainly to dicots in which a severely wrinkled phenotype, reduced internode distances, and abnormal reproductive development were commonly observed. In this work, we analyzed the effects associated with the expression of this gene in a new genetic context: the forage grass genome. Transgenic P 35S •rolA plants of blue grama grass (Bouteloua gracilis) were obtained by a biolistic approach employing embryogenic chlorophyllic cells as the target material. Four independent transgenic lines with regeneration capacity were recovered, which showed stable integration of this transgene as demonstrated by polymerase chain reaction and Southern blot hybridization. Growth of the rolA-transformed lines under greenhouse conditions provided evidence for a new biotechnological application for the rolA gene in plants, namely, the improvement of biomass production in forage grasses. Additionally, we described here a new phenotypic marker (referred to here as the "hairy embryo" syndrome) that can be instrumental for the early identification of transformation events when transforming grasses with this gene.
Biologia plantarum, 2008
A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared to the whole plasmid. The Gfp expression was stable up to the T 2 generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation in bentgrass with possible applications to other plant species.
Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation
Plant Cell Reports, 2006
Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4-5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10-12 weeks after Agrobacteriummediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass.
Agrobacterium-mediated genetic transformation of plants: biology and biotechnology
Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.
Factors Affecting Agrobacterium-mediated Transformation of Plants
Plant transformation technology has become a versatile platform for cultivar improvement as well as for studying gene function in plants. The development of an efficient method for genetic transformation is a prerequisite for the application of molecular biology to the improvement of a given crop species. Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains and technologies currently used for the successful transformation of numerous plant species. Many factors influencing Agrobacterium-mediated transformation of plants have been investigated and elucidated. These factors include bacterial strains and cell density, plant species and genotype, plant growth regulators and antibiotics, explant, explant wounding, light and temperature. Before attempting stable transformation of any new species, it is useful to optimize the factors influencing transformation efficiency, as this can reduce future costs in labor and materials. The studies of such factors hold great promise for the future of plant biotechnology and plant genetic engineering as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants. Here, I review some of the main factors that influence Agrobacterium-mediated genetic transformation and discuss their possible roles in this process.
Production of Transgenic Plants via Agrobacterium-Mediated Transformation in Liliaceous Ornamentals
Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues Vol. II, 2006
Studies on the Agrobacterium-mediated production of transgenic plants in several Liliaceous ornamentals, Lilium spp., Agapanthus spp., Muscari armeniacum and Tricyrtis hirta are described. Different strains of A. tumefaciens were used, all of which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII) gene, the intron-containing ȕ-glucuronidase (GUS) gene, and the hygromycin phosphotransferase (HTP) gene in the T-DNA region. Utilization of organogenic or embryogenic calluses as a target material for transformation and acetosyringone (AS) treatment during inoculation and/or co-cultivation with Agrobacterium were found to be critical for successful production of transgenic plants in Liliaceous ornamentals. Following transfer of co-cultivated organogenic or embryogenic calluses onto hygromycin-containing media, several hygromycin-resistant (Hyg r) tissues were obtained, and complete plants were subsequently developed from these tissues. Most of the plants were verified to be transgenic plants by GUS histochemical assay and PCR analysis. For Lilium 'Acapulco', A. praecox ssp. orientalis 'Royal Purple Select' and M. armeniacum 'Blue Pearl', Southern blot or inverse PCR analysis revealed the integration of 1-5 copies of the transgene into the genome of transgenic plants, but most of them had 1 or 2 copies. Agrobacterium-mediated transformation systems thus established may be useful as a tool for molecular breeding as well as molecular biological studies.