Localization of Persisting Agrobacteria in Transgenic Tobacco Plants

Matzk, Anja

doi:10.1094/mpmi-9-0373

Cited by 41 publications

(22 citation statements)

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“…1). This is in contrast to the findings of Matzk et al (1996) and Barrett et al (1997) who isolated A. tumefaciens from 12-month-old transgenic tobacco plants and 24-week-old Brassica shoots, respectively. Possible explanations for this difference are that the Agrobacterium strains, the concentration of the inoculum and the washing method after co-cultivation for elimination were not the same for any of the species examined.…”

Section: Embryogenic Tissue Cultured For Three Months To Two Years Afcontrasting

confidence: 99%

“…Our results differ from a study of transgenic soilgrown tobacco in which A. tumefaciens was found predominantly in the stems and roots after 3-6 months (Matzk et al, 1996). These findings may be a predictor for the location of A. tumefaciens in other transgenic plants and are in agreement with evidence for Agrobacterium species, which exist as non-pathogenic endophytes, being localized to the roots or root cortex (Hallman et al, 1997;Yang et al, 1999).…”

Section: Transgenic Trees Grown In the Gmo Greenhouse For Two To Fourcontrasting

confidence: 73%

“…Developing somatic embryos or germinating plantlets, which had been produced in vitro, were evaluated after three or four and a half months, respectively. To increase the chance of detecting A. tumefaciens that might be present but not actively growing, it was necessary to apply an enrichment culture, derived from Matzk et al (1996). Briefly, 1 g of transgenic tissue was macerated in 10 ml of YEP liquid medium and grown at 27°C without antibiotics for seven days.…”

Section: In Vitro-grown Embryogenic Tissue Developing Somatic Embryomentioning

confidence: 99%

“…Unfortunately, there are disproportionately few publications investigating the survival of residual A. tumefaciens in plant tissues after transformation, relative to those on A. tumefaciens-mediated transformation per se. Of these few studies, Matzk et al (1996) showed that A. tumefaciens was detectable in transgenic tobacco grown in vitro for as long as one year after transformation. They also found that A. tumefaciens persisted in transgenic tobacco plants that had been in soil for 3-6 months, and this appears to be the only study of A. tumefaciens persistence in transgenic plants grown ex vitrum.…”

Section: Introductionmentioning

confidence: 99%

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Persistence ofAgrobacterium tumefaciensin transformed conifers

Charity

Klimaszewska

2005

Environ. Biosafety Res.

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Previous studies have shown that the widely used plant transformation vector Agrobacterium tumefaciens can persist in genetically engineered plants in vitro and in transgenic greenhouse-grown plants, despite the use of counter-selective antibiotics. However, little is known regarding Agrobacterium persistence in tree species. To understand the kinetics of A. tumefaciens decline and persistence in transformation experiments, we assayed for the presence of A. tumefaciens in spruce and pine embryogenic tissue for up to 10 weeks posttransformation. The A. tumefaciens populations declined rapidly in the first five days post-cocultivation but generally declined more slowly in pine, relative to spruce. No bacteria were detected in spruce embryogenic tissue beyond four weeks after cocultivation, however in pine there were ~100 colony forming units per g tissue at 10 weeks post-cocultivation. We present evidence that the detection limit for PCR using virD 2 primers to detect A. tumefaciens in a background of pine needle DNA was approximately 10 9 -10 10 A. tumefaciens cells per g of tissue. We also assayed for A. tumefaciens in transgenic pine and spruce embryogenic tissue and from needles, branches, stems and roots of transformed plants, up to four years post-inoculation. Occasionally A. tumefaciens was detected in embryogenic tissue up to 12 months post-inoculation. A. tumefaciens was never detected in cultured embryogenic tissue more than twelve months after inoculation, nor in developing somatic embryos or germinating plantlets, nor any of the parts of greenhouse-grown plants. From these data we conclude that if A. tumefaciens persists in transgenic conifers, it does so beneath our ability to detect it.

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Section: Embryogenic Tissue Cultured For Three Months To Two Years Afcontrasting

confidence: 99%

Section: Transgenic Trees Grown In the Gmo Greenhouse For Two To Fourcontrasting

confidence: 73%

Section: In Vitro-grown Embryogenic Tissue Developing Somatic Embryomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Persistence ofAgrobacterium tumefaciensin transformed conifers

Charity

Klimaszewska

2005

Environ. Biosafety Res.

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“…Ecological risks of biological vectors persisting in transgenic plants are nil for direct DNA transfer but high for Agrobacteriummediated transfer (62,63).…”

Section: Discussionmentioning

confidence: 99%

Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Kohli

Leech

Vain

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

245

130

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Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a twophase integration mechanism. In the ''preintegration'' phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and͞or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.Efficient genetic manipulation of major crops, including cereals, has been made possible by transfer of foreign DNA into the host genome through direct DNA transfer procedures such as particle bombardment (1-3). Recently, Agrobacterium-mediated transformation of rice (4), maize (5), and barley (6) has been reported.Much research has been undertaken to understand the mechanism of Agrobacterium-mediated transfer and integration of foreign DNA into plant genome (7). However, little is known about equivalent mechanisms for foreign DNA integration into plants generated through bombardment or other direct DNA transfer methods. Understanding the mechanism of integration of transgenes through direct DNA transfer is important in elucidating, at the plant cell level, events that characterize the process of DNA integration, without the bacterial gene products involved in Agrobacterium-mediated transformation. This understanding will be crucial to achieve the targeted gene delivery. As most transgenic plants (certainly all cereals) in field trials and on the market have been generated through particle bombardment, it assumes more importance.Transgenic plants generated either through Agrobacteriummediated transformation or direct DNA transfer share a common feature, that of transgenic loci frequently consisting of multiple copies of the transgenes. This varies from species to species, and in general, it is accepted that transgenic solanaceous plants generated through Agrobacterium-mediated transformation contain lower transgene copy numbers and simpler integration patterns (8). In species such as rice, maize, and barley (...

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Plant Transformation Technology:Agrobacterium‐Mediated Transformation

Komari

Ishida

Hiei

2004

Handbook of Plant Biotechnology

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The soil phytopathogen Agrobacterium tumefaciens induces tumours, known as crown galls, mainly on dicotyledonous plants. Such tumours are generated by a complex, multi‐step transformation process. Another species, A. rhizogenes , causes hairy root disease in higher plants via an identical process. Agrobacterium has been utilised for the transfer of genes to dicotyledonous plants. Now, monocotyledonous plants are routinely transformed by Agrobacterium despite the fact that these plants, including important cereals, were thought until recently to be outside the range of this technology. Most of the common transformation methods heavily depend on tissue culture technology and we refer to such methods as in vitro transformation. The only case of a routine tissue culture free method is the ‘ in planta ’ transformation of Arabidopsis thaliana . Numerous critical factors are involved in both approaches. In transformation in vitro , key factors include choice of vectors and bacterial strains, types of plant tissues to be infected, procedures of preparation the tissues, protocols of infection and co‐cultivation, methods for subsequent culture and selection of transformed cells, antibiotics to remove infecting bacteria, techniques for regeneration of transgenic plants and genotypes of plants. It is our opinion that the type and quality of the starting material is the most important one among them. The capacity to serve as a host plant for crown gall disease is no longer a prerequisite for a host of vector systems based on Agrobacterium . The real prerequisite for in vitro transformation is the availability of technology for dedifferentiation of tissues and regeneration of plants in a given species. On the other hand, the biological processes involved in in planta transformation are yet to be elucidated. Recent evidence suggested that ovules are the primary target, and further basic understanding is likely to help extend the number of species transformed by the method. The plant species that are routinely transformed by Agrobacterium are expanding rapidly. Some gymnosperms, several forest trees and fruit trees, various legumes, and cereal and non‐cereal monocotyledons, which were once considered very recalcitrant, are now in a long list of transformable plants. Arabidopsis , tobacco and rice are the top three species that were transformed during the last two years. Although it is only half a dozen years since the current procedure of rice transformation mediated by Agrobacterium was published, the economic importance and the accumulation of genomic information has made rice the species of focus in plant biotechnology. The advantages of Agrobacterium ‐mediated transformation include the transfer of pieces of DNA with defined ends and minimal rearrangement, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes and the high quality and fertility of transgenic plants. However, transformation does not always produce such ‘clean’ events. Formation of repeats of transgenes, certain rearrangements, integration of non‐target DNA segments and unstable expression of transgenes are among the complications. Although the majority of transgenic plants usually appear ‘good’ in a particular test, accumulation of ‘dropped’ plants is significant after multiple rounds of characterisation and screening. Therefore, further improvement in each of steps is highly desired. Application of the gene transfer mediated by Agrobacterium is further expanding. Transient expression of genes delivered by Agrobacterium is now a useful tool in the study of promoters and gene function. Vectors specifically designed to carry very large segments of DNA have been developed and extensively tested. Insertional mutagenesis by DNA transferred by Agrobacterium is a routine technique in genomics study in Arabidopsis and rice. Methods for targeted integration of transgenes to genomes of higher plants have been drawing considerable attention. Various technologies for the production of ‘selection marker free’ transgenic plants are now in place for better public acceptance of biotechnology products.

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Localization of Persisting Agrobacteria in Transgenic Tobacco Plants

Cited by 41 publications

References 0 publications

Persistence ofAgrobacterium tumefaciensin transformed conifers

Persistence ofAgrobacterium tumefaciensin transformed conifers

Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Plant Transformation Technology:Agrobacterium‐Mediated Transformation

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