Marker For Genetic Transformation Research Articles

RNAi-induced knockdown of white gene in the southern green stink bug (Nezara viridula L.)

The southern green stink bug (SGSB) Nezara viridula L. is one of the most common stink bug species in the United States and can cause significant yield loss in a variety of crops. A suitable marker for the assessment of gene-editing tools in SGSB has yet to be characterized. The white gene, first documented in Drosophila, has been a useful target to assess the efficiency of introduced mutations in many species as it controls pigmentation processes and mutants display readily identifiable phenotypes. In this study we used the RNAi technique to investigate functions and phenotypes associated with the white ortholog in the SGSB and to validate white as a marker for genetic transformation in this species. This study revealed that white may be a suitable marker for germline transformation in the SGSB as white transcript knockdown was not lethal, did not impair embryo development and provided a distinguishable phenotype. Our results demonstrated that the white ortholog in SGSB is involved in the pathway for ommochrome synthesis and suggested additional functions of this gene such as in the integument composition, management of hemolymph compounds and riboflavin mobilization.

The usage of snapdragon Delila (Del) gene as a visible selection marker for the antibiotic-free transformation system

This study was conducted to assess the suitability of the Delila gene (Del) from snapdragon for use as a visible selectable marker, under the control of the cauliflower mosaic virus (CaMV) 35S promoter, to develop a plant genetic transformation system that helps to avoid using antibiotic- or herbicide-resistance genes, such as the gene for resistance against kanamycin or PPT. Following transformation, tobacco shoots showing red coloration always contained the Del gene, which was confirmed by PCR analysis. No chimerism or ploidy variation was observed during genetic transformation. In addition, the integration ratio of the transgene to the T1 progeny was 3:1, following typical Mendelian fashion. By anthocyanin analysis, the plants containing the Del gene were shown to have 80 times higher anthocyanin content than the control plants. Thus, we conclude that strong anthocyanin accumulation, as a result of the snapdragon Del gene, can be used as a visible selectable marker for genetic transformation in the tobacco plant, replacing the use of antibiotic- or herbicide-resistance genes.

Studies on the sensitivity of Spirulina platensis to antibiotics and herbicide: relationship with selectable markers for genetic transformation

The cyanobacterium Spirulina platensis was tested for sensitivity to 6 antibiotics: kanamycin (Km), neomycin (Neo), geneticin (G418), ampicillin (Ap), chloramphenicol (Cm), hygromycin (Hm) and to 1 herbicide: L-phosphinothricin (PPT), all of which are frequently applied as selectable markers in plant gene engineering. Growth tests, carried out at different concentrations of the agents above, showed that S. platensis was sensitive to Hm, Cm and PPT, while highly resistant to Km, Neo, G418 and Ap, indicating that Hm, Cm and PPT were potent inhibitors of S. platensis growth and able to be employed as selectable markers for further transformation studies for this alga.

A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius.

We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5'-phosphate decarboxylase (URA3) or orotidine-5'-pyrophosphate (URA5). Uracil auxotrophs of S. alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S. cerevisiae. A transformation frequency of approximately 10(4)/micrograms DNA was obtained, which is tenfold higher than results described in either reports. Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable. The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue. The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid.