Advances in the Transformation of Cyclamen persicum Mill. Through Direct Regeneration Based on an Optimized Kanamycin Selection Scheme.

Gene transfer technology has great value in ornamental plants toward the generation of varieties with new ornate characteristics. In the previous studies through the transformation of cyclamen, hygromycin was mainly used as a selective marker. However, there have been some drawbacks associated with hygromycin usage as a selecting agent. Therefore, in the current study, the optimization of kanamycin concentration in the regeneration media has been considered. Subsequently, the plant transformation using three different in vitro explants from three Cyclamen persicum cultivars using three Agrobacterium tumefaciens strains has been examined. Accordingly, the optimal kanamycin concentrations for regeneration from root and leaf explants were determined as 10 mg/L and for microtuber explants as 30 mg/L. The successful gene transformation in the antibiotic-resistant shoots were examined by PCR and UV-equipped microscopes. The gfp reporter gene transfer resulted in the highest efficiency of transformation (60%) to date, from the leaf explants of cv. Pure White inoculated with Agrobacterium tumefaciens strain LBA4404. In contrast, the lowest gene transfer efficiency (25%) was observed in root explants of cv. Dark Violet and cv. Neon Pink inoculated with strains GV3101 and AGL-1, respectively. The results of the current project are expandable to the subsequent investigations of Cyclamen persicum transformation.

Recovery of virus-free Almond (Prunus dulcis) cultivars by somatic embryogenesis from meristem undergone thermotherapy.

One of the world's main horticulture problems is the contamination of fruit trees with a variety of plant diseases, especially viral and pseudo-viral diseases. Due to the non-sexual propagation of the trees, these diseases have been transmitted to different parts of the world. The main aim of this study was to obtain a new effective method for virus elimination from almond cultivars, which was performed in two phases. In the first phase, we tested various almond cultivars with ELISA and RT-PCR. The results showed the infection of mother plantlets. So, three types of in vitro thermotherapy treatments were performed on infected plants to make them virus-free. The plantlets obtained from 0.5 mm meristem treated with the first type of thermotherapy (TH1: 8 h at 27 °C and 16 h at 38 °C for 18 days) showed the highest percentage of elimination of ApM, ACLS and TRS viruses. In the second phase, meristems were cultured on MS medium containing 0, 0.5, 1 and 2 mg/L 2,4-D with 1 mg/L TDZ and after two weeks, thermotherapy treatments were performed. The results showed, combining three methods of thermotherapy (TH1), meristem culture and somatic embryogenesis induction from meristem on MS medium supplemented with 0.5 mg/L 2,4-D and 1 mg/L TDZ is the most effective and safe technique for virus eradication without meristem size challenges. The samples that were diagnosed as virus-free were proliferated in temporary immersion bioreactor systems, and rooted to be used for later propagation and establishment of mother healthy orchards.

Boosting riboswitch efficiency by RNA amplification.

Riboswitches are RNA sensors that regulate gene expression in response to binding of small molecules. Although they conceptually represent simple on/off switches and, therefore, hold great promise for biotechnology and future synthetic biology applications, the induction of gene expression by natural riboswitches after ligand addition or removal is often only moderate and, consequently, the achievable expression levels are not very high. Here, we have designed an RNA amplification-based system that strongly improves the efficiency of riboswitches. We have successfully implemented the method in a biological system for which currently no efficient endogenous tools for inducible (trans)gene expression are available: the chloroplasts of higher plants. We further show that an HIV antigen whose constitutive expression from the chloroplast genome is deleterious to the plant can be inducibly expressed under the control of the RNA amplification-enhanced riboswitch (RAmpER) without causing a mutant phenotype, demonstrating the potential of the method for the production of proteins and metabolites that are toxic to the host cell.