Towards genetic engineering of maritime pine (Pinus pinaster Ait.) (original) (raw)
Related papers
Towards genetic engineering of maritime pine ( Pinus pinaster Ait
Annals of Forest Science, 2002
Using our improved protocols for somatic embryogenesis in Pinus pinaster, transgenic tissues and plantlets were recovered after microprojectile bombardment (biolistic) or cocultivation of embryonal-suspensor masses (ESM) with Agrobacterium tumefaciens. Transformation experiments were carried out with selectable hpt gene (hygromycin B resistance) and reporter gus gene (β-glucuronidase activity). With both methods, hygromycin was shown to be an effective selective agent of transformed cells within 4-19 weeks. The mean number of hygromycin-resistant lines expressing gus per gram ESM subjected to DNA transfer, ranged from 7.0 to 8.5 using biolistic and 0 to 67.3 during Agrobacterium experiments. Mature somatic embryos obtained from some transformed lines were converted into plantlets and grown in the greenhouse. The whole process (from transformation to plant acclimatisation) could be completed within only 12 months. The transgenic state of ESM, somatic embryos and plants was confirmed by histochemical GUS assays and molecular methods.
1999
A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and β-glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized.
Agrobacterium tumefaciens-Mediated Genetic Transformation of Pinus kesiya Royle ex Gord (Khasi Pine
This study highlights, for first time, an Agrobacterium-mediated gene transfer method for the genetic improvement of Pinus kesiya. The genetic transformation of P. kesiya has been limited by difficulty in selection efficiencies and a low transformation frequency. Embryo-genic cultures were established from zygotic embryos according to our previous protocol (Malabadi et al. 2005). During transformation events, rapidly growing embryogenic tissue of three genotypes were co-cultivated with disarmed A. tumefaciens strain EHA105 fused with a binary vector pBI121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the-glucuronidase (uidA) reporter gene, was used in the transformation studies. All the transgenic lines exhibited very low maturation potential compared to the control. GUS activity was used to monitor transient expression of the uidA gene and to further test lines selected on kanamycin-containing medium. The integration of one of the transgenes, nptII, was confirmed by PCR followed by Southern and Northern blot analyses. Agrobacterium-mediated gene transfer was found to be a very useful technique for large-scale generation of transgenic P. kesiya, and may prove useful for other recalcitrant conifer species.
Planta, 2001
Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymer...
Stable and Consistent Agrobacterium-Mediated Genetic Transformation in Pinus roxburghi (Chir Pine
This paper highlights an Agrobacterium tumefaciens-mediated transformation protocol, developed for embryogenic cell cultures derived from vegetative shoot apices of mature, 14 years-old trees of Pinus roxburghii. The plasmid pBI121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the-glucuronidase (uidA/GUS) reporter gene, was used as a binary vector. Transformation frequencies were dependent on the species, genotype and post-cocultivation procedure. The highest transformation efficiency was obtained in the embryogenic line PR105 (37 transformed lines/g fresh wt) than with the embryogenic lines PR11 and PR521. The transgenic state of the embryogenic tissue was initially confirmed by histochemical GUS assay. Stable integration of the nptII gene in the plant genome of P. roxburghii was confirmed by polymerase chain reaction (PCR), Southern and Northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in chir pine, and that this system would provide an opportunity to transfer economically important genes into other genotypes of P. roxburghii.
We established for the first time an Agrobacterium-mediated genetic transformation system for the Himalayan blue pine. Embryogenic tissue derived from vegetative shoot apices of mature trees of Pinus wallichiana were inoculated with A. tumefaciens strain EHA105 fused with a binary vector pBI121. The plasmid pBI121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the-glucuronidase (uidA) reporter gene, was used in the transformation studies. GUS activity was used to monitor transient expression of the uidA gene and to further test lines selected on kanamycin-containing medium. The integration of the transgene (nptII) was confirmed by PCR followed by southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system could be established in P. wallichiana. This provides an opportunity to transfer economically important genes into Himalayan blue pines.
Plant Cell Tissue and Organ Culture, 2006
The effects of antibiotics commonly used in Agrobacterium-mediated transformation were studied on Pinus pinaster tissues. Embryogenic tissue growth from three embryogenic lines and adventitious bud induction from cotyledons from three open-pollinated seed families were analysed. Cefotaxizme, carbenicillin and timentin commonly used for Agrobacterium elimination, at concentrations of 200-400 mg l -1 did not inhibit the embryogenic tissue growth on filter paper nor as clumps. Adventitious bud induction and bud number were significantly reduced for one of the tested families when using 400 mg l -1 cefotaxime or timentin. The selection agent kanamycin significantly inhibited growth of embryogenic tissue on filter paper in all the embryogenic lines and concentrations tested (20-50 mg l -1 ).
Plant Cell Reports, 1994
A biolistic particle delivery system was used to transform nodal segments of teak (Tectona grandis). The introduced DNA contained a β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes each fused to the CaMV 35S promoter. Merely 70% from the bombarded nodal segments survived at the first screening and the survival rate was further reduced to 15% following the second screening. Subsequently, only 8% of the plants derived from bombarded nodal segments reached the nursery stage. Expression of the uidA gene was detected histochemically in putative transformed tissues and subsequently in regenerated plants. The presence of the uidA gene and its integration into the teak genome was demonstrated using PCR and PCR-Southern hybridization. PCR analyses using uidA primers showed that 59% of the putative transformed plants selected for hygromycin resistance were transformed with the gene encoding β-glucuronidase. The expression of uidA gene in transformed plant was again confirmed by RT-PCR analysis. This is the first report of a method to transform genetically teak.
An Agrobacterium-mediated transformation procedure was developed to transform the mature embryo from Pinus monticola (Dougl. ex D. Don) seeds with two binary vectors containing the reporter gene encoding the green fluorescent protein (GFP) or the-glucuronidase protein (GUS), respectively. More than 1000 embryos from independent transformation events were tested for different western white pine seed families. Selection of kanamycin-resistant callus tissues showed that survival rates varied from 33 to 48% in different independent experiments. Transgenic callus tissues survived and continued to grow on the medium with kanamycin (25 g/mL), whereas non-transgenic callus, regenerated from the embryos of the same seed family, died within 12 weeks. Integration and expression of the introduced reporter gene was confirmed in transgenic western white pine calli by GUS-staining analysis or microscopic observation of GFP fluorescence. Rates for stable reporter gene expression ranged from 2.9 to 6.5% for all embryos co-cultured with Agrobacterium. Our protocol has enabled the routine transformation of western white pine, a species that was previously difficult for gene manipulation. To our knowledge, this is the first report on genetic engineering of this conifer. Our results demonstrate that transgenic gene expression in western white pine is a feasible option for genetic improvement of this valuable conifer as well as for investigating its molecular interactions with the fungal pathogen Cronartium ribicola (J.C. Fisch.).