Stable transformation of embryogenic tissues of Pinus nigra Arn. using a biolistic method.
ABSTRACT The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l(-1). Resistant embryogenic tissue recovery required 10-12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained.
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ABSTRACT: In developing and developed nations, plant cell culture systems are used to supply desirable compounds in lieu of chemical synthesis or natural extraction. When plant cell culture systems are unable to meet commercial demand, metabolic engineering offers a method to increase yields. However, to benefit from metabolic engineering approaches, effective transient transformation methods are required to rapidly identify and characterize key regulatory genes before intensive, time-consuming stable transformation efforts can proceed. This paper describes a particle bombardment-mediated transient transformation system for Taxus spp. in cell culture. Optimal parameters were established for the T. cuspidata cell line P991 and the T. canadensis cell line CO93D, resulting in reliable, efficient, transient expression of the firefly luciferase gene under control of the constitutive CaMV 35S promoter. Multiple bombardments and larger gold microcarriers (1.6 vs 1.0 microm in diameter) were particularly effective in increasing luciferase activity and in reducing variation among replicates. This particle bombardment-mediated transformation system was also shown to be capable of transiently expressing the DsRed and beta-glucuronidase reporter genes under the control of the maize ubiquitin and CaMV 35S promoters, respectively. With the ability to transiently transform Taxus spp. cell cultures using a variety of promoters and reporters, characterization of genes related to paclitaxel accumulation in culture can now proceed.Biotechnology Progress 08/2007; 23(5):1180-5. DOI:10.1021/bp0700307 · 1.88 Impact Factor
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ABSTRACT: Advances in conifer tissue culture and genetic transformation offer new opportunities in the field of genetic engineering. Genetic transformation is an important tool for breeders because it allows them to introduce valuable genes that might otherwise be difficult to integrate into elite genotypes. Combined with mass vegetative propagation, such as somatic embryogenesis, regeneration of transgenic conifers could result in accelerated tree improvement. Genetic transformation requires the development of an efficient gene delivery system. Extensive work has been done in Pinus spp. to improve transformation methods, either by DNA-coated particle bombardment or by co-culture with Agrobacterium tumefaciens. This review describes the most recent progress made in genetic transformation in the genus Pinus, with emphasis on four important forest and plantation species grown in Europe (P. pinaster), New Zealand, Australia and South America (P. radiata), and North America (P. taeda and P. strobus). The biosafety issues associated with potential deployment of transgenic pine varieties in commercial forestry are highlighted.