Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures

Charity, Julia A.; Holland, L.; Grace, Lynette J.; Walter, Christian

doi:10.1007/s00299-004-0851-6

Cited by 34 publications

(19 citation statements)

References 30 publications

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“…No A. tumefaciens was isolated from any of the un-inoculated controls in this or the previous experiment (data not shown). A. tumefaciens levels decreased more slowly in radiata pine than in black spruce, probably because the black spruce culture medium contained both cefotaxime and Timentin (Klimaszewska et al, 2001), whereas radiata pine was maintained only on Timentin (Charity et al, 2005). Radiata pine could not be transferred to a medium containing cefotaxime because this antibiotic is known to be detrimental to the health of other P. radiata explants (such as cotyledons) (Holland et al, 1997).…”

Section: Quantification In Embryogenic Tissue 1-10 Weeks Post-inoculamentioning

confidence: 99%

“…1A). One explanation is that radiata pine is washed through a Buchner funnel with liquid medium (Charity et al, 2005) whereas for the other species, the tissue is resuspended and collected on a Buchner funnel (Klimaszewska et al, 2001). Seven days post-inoculation, all tissue from all species appeared to be bacteria-free by visual inspection but culturing macerated tissue followed by serial dilution showed that A. tumefaciens populations were ~10 4 colony forming units CFU.g -1 tissue for both spruce species, 10 5 CFU.g -1 tissue for white and radiata pine and 10 6 CFU.g -1 tissue for maritime pine (Fig.…”

Section: Quantification 0-7 Days Post-inoculationmentioning

confidence: 99%

“…All embryogenic tissue was inoculated with A. tumefaciens (strain C58; pMP90) at a concentration of ~10 9 cells per ml. After cocultivation, radiata pine was washed on a Buchner funnel under gentle vacuum with ½EMS3 liquid medium to remove excess A. tumefaciens (Charity et al, 2005). For the other species, embryogenic tissue from five plates (at a time) were dislodged by manual shaking into an Erlenmeyer flask (250 ml) with 100 ml mLV medium and subsequently collected on new filter papers (Whatman No.…”

Section: Plant Transformationmentioning

confidence: 99%

“…Embryogenic tissue of two spruce species, Picea mariana (black spruce) and P. glauca (white spruce) and three pine species, Pinus pinaster (maritime pine), P. strobus (eastern white pine) (Klimaszewska et al, 2001) and P. radiata (radiata pine) (Charity et al, 2005) were inoculated, co-cultivated and washed according to published methods. The supervirulent strain A. tumefaciens strain C58 (pMP90) (Koncz and Schell, 1986), containing the binary vector pRGR1 carrying the uidA gene (without an intron) driven by 2 × 35S promoter and terminated by the 35S terminator (R. Rutledge, Canadian Forest Service, unpublished) was used.…”

Section: Plant Transformationmentioning

confidence: 99%

“…While A. tumefaciens-mediated transformation methods have been developed for a number of important forest species (e.g. Charity et al, 2005;Klimaszewska et al, 2001;Le et al, 2001;Levée et al, 1999;Trontin et al, 2002), there are currently no published data on the persistence of A. tumefaciens in transgenic trees or any data evaluating the persistence of A. tumefaciens in any soil-grown plants for longer than six months. However, this sort of information is an important pre-requisite for field release of transgenic plants from a regulatory standpoint.…”

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: Quantification In Embryogenic Tissue 1-10 Weeks Post-inoculamentioning

confidence: 99%

Section: Quantification 0-7 Days Post-inoculationmentioning

confidence: 99%

Section: Plant Transformationmentioning

confidence: 99%

Section: Plant Transformationmentioning

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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Recent Advances in Plant Transformation

Barampuram

Zhang

2010

Methods in Molecular Biology

109

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Plant genetic engineering has become one of the most important molecular tools in the modern molecular breeding of crops. Over the last decade, significant progress has been made in the development of new and efficient transformation methods in plants. Despite a variety of available DNA delivery methods, Agrobacterium- and biolistic-mediated transformation remain the two predominantly employed approaches. In particular, progress in Agrobacterium-mediated transformation of cereals and other recalcitrant dicot species has been quite remarkable. In the meantime, other transgenic-enabling technologies have emerged, including generation of marker-free transgenics, gene targeting, and chromosomal engineering. Although transformation of some plant species or elite germplasm remains a challenge, further advancement in transformation technology is expected because the mechanisms of governing the regeneration and transformation processes are now better understood and are being creatively applied to designing improved transformation methods or to developing new enabling technologies.

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