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.

Immune analysis of cry1Ab-genetically modified potato by in-silico analysis and animal model.

The safety of feeding transgenic potato (Solanum tuberosum), resistant to the potato tuber moth, to Wistar rats was examined from an immunological perspective. The genetically modified potato (GMP) was harbouring cry1Ab and nptII genes as target and selectable marker genes, respectively. In-silico analysis reconfirmed that Cry1Ab and NPTII protein sequences have no significant homology to known toxins or known allergens. The Wistar rats were fed a diet containing 20% GMP or its parental control, non-GMP (NGMP), for 90 days. The consumption of GMP food did not affect the growth rate, food intake, food efficiency, and general health status of the rats. There were no significant differences in the serum concentrations of immunoglobulin A (IgA), IgG, IgM, interleukin 6 (IL-6), and IFN-γ between GMP and NGMP-fed rats. Based on such data, it is concluded that the transgenic potato had no adverse effect on immunity functions of Wistar rats.

Genetic transformation of date palm (Phoenix dactylifera L. cv. 'Estamaran') via particle bombardment.

In this study, an efficient transformation system for gene delivery in date palm was established. The effects of different physical and biological parameters were optimized for transient transformation of uidA gene in somatic embryos of Estamaran cultivar. The tissues were bombarded with constructs harboring the uidA gene driven by CaMV 35S or rice Act1 promoter. Efficiency of expression was estimated by comparison of the number of blue spots resulted from GUS assay. Optimal transient expression was observed when explants were precultured on a media containing 0.4 M mannitol with air desiccation and bombarded at acceleration pressure of 1,350 psi, target distance of 6 cm with gold particles size of 0.6 µm which coated with 2.5 µg of DNA and at chamber vacuum pressure of 28 inHg. Significantly higher expression levels were obtained in tissues when the construct having the Act1 promoter was employed. After bombardment, somatic embryos were transferred to the regeneration media containing MS basal salts supplements with 3 mg/l 2ip, 40 mg/l adenine, 1 mg/l 2,4-D, 30 g/l sucrose and 3 g/l activated charcoal. Regenerated plantlets were checked by PCR using gene-specific primers. About 16 % of the plantlets were reported to be stably transformed. Southern analysis of genomic DNA from transformed plants showed that 1-2 gene (uidA) copies were integrated and GUS-negative plants did not contain any transgene. Achievement of these data considered as the first report of its kind is believed to facilitate transfer of desirable traits in date palm.

Human granulocyte colony-stimulating factor (hG-CSF) expression in plastids of Lactuca sativa.

BACKGROUND: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. METHODS: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. RESULTS: hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. CONCLUSIONS: This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.