The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

Acquisition of an Agrobacterium Ri Plasmid and Pathogenicity by Other α-Proteobacteria in Cucumber and Tomato Crops Affected by Root Mat

ABSTRACT Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring α-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium. An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring α-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).

Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria

ABSTRACT Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Emr) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 × 104 and 1.0 × 104 CFU/0.5 μg of DNA, with standard deviations of 0.54 × 104 and 0.32 × 104, for shsp and Emr selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 × 104 and 3.8 × 103 CFU/0.5 μg of DNA, with standard deviations of 0.63 × 104 and 3.48 × 103, for shsp and Emr selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.

Repeatability of a Real-Ear-to-Coupler Difference Measurement as a Function of Age

For a given individual, the applicability of an average real-ear-to-coupler transfer function in the fitting of hearing aids is of limited utility, because the acoustical properties of individual ears may differ substantially from average transformation values (Feigin, Kopun, Stelmachowicz, & Gorga, 1989; Fikret-Pasa & Revit, 1992). It has been suggested, therefore, that individual real-ear-to-coupler difference (RECD) measures should be obtained whenever possible and applied within the fitting process (Hawkins, 1992; Nelson Barlow, Auslander, Rines, & Stelmachowicz, 1988). The purpose of this study was to investigate the repeatability of a specific RECD measurement procedure that has been developed for clinical application with young children (Moodie, Seewald, & Sinclair, 1994). The test-retest reliability of this procedure is reported for 10 adults and 90 children in the birth-to-7-year age range.

Precise Incubation Period for the Agrobacterium- mediated Transformation Efficiency in Potato (Solanum tuberosum L.) cvs. Cardinal and Atlas

Precise incubation period facilitates significant response to Agrobacterium- mediated genetic transformation of potato (Solanum tuberosum L.) cvs. Atlas and Cardinal. The protocol yielded an average transformation rate of 58.4% for internodal explants compared to 38% for leaf explants in Atlas while 47.6% for intenodal explants as against 7.6% for leaf explants in Cardinal. Highest survival rate and transient GUS activity were shown by Atlas and Cardinal at 45 min of incubation with Agrobacterium. The plants were analyzed histochemically for GUS activity in their leaves and internodes. All these analysis indicated that each independently selected and regenerated plants of Atlas and Cardinal were GUS positive and transient. Key words: Transformation, Incubation period, Potato cultivars D.O.I. 10.3329/ptcb.v19i2.5440 Plant Tissue Cult. & Biotech. 19(2): 227-235, 2009 (December)