A positive experience in applying the biolistic approach to potato varieties Aksor and Nevskiy

The method of biological ballistics (biolistic transformation, genetic bombardment) of plants is one of the most modern methods used for direct gene transfer into plant cells. The main advantages of this method include the ability to simultaneously incorporate several target genes into the plant genome, carry out transfer without unnecessary agrobacterial parts and plasmid DNA sequences, and the short time needed to produce transgenic cells. For different plant objects, the efficiency of obtaining transgenic plants by the ballistic method varies from 1 to 3 %. For potato plants, the transformation efficiency is quite low at the moment and the selection of optimal conditions for biolistics is one of the pressing issues of practical biotechnology. This article presents a successful experience of introducing two genes of interest into two potato varieties using the biolistic approach. The results of biolistic transformation experiments are presented for two types of explants: potato internodes and calli of the varieties Aksor and Nevskiy. Of the 862 explants used for transformation, 56 regenerated plants were obtained. PCR screening of transformants revealed one plant with the insertion of the chitinase gene, one with the insertion of the endo-β-1,3-glucanase gene, and co-transformation by both genes was confirmed in four regenerants. The average transformation efficiency for potato explants was 0.7 %. A high number of regenerants (56) as opposed to a low number of transformants (6) reflects an attempt to increase the number of regenerants by using a lower concentration of the selective agent (kanamycin). Although this approach requires more effort, it can be used to produce potato lines with integrated genes of interest for further use in crop breeding. The lines of potato obtained in the current study by introducing two genes associated with the plant response to fungal pathogens will be further assessed for their resistance to fungal diseases and, if successful, will be used in potato crop breeding.

Development and characterization of Triticum turgidum–Aegilops umbellulata amphidiploids

The U genome of Aegilops umbellulata is an important basic genome of genus Aegilops. Direct gene transfer from Ae. umbellulata into wheat is feasible but not easy. Triticum turgidum–Ae. umbellulata amphidiploids can act as bridges to circumvent obstacles involving direct gene transfer. Seven T. turgidum–Ae. umbellulata amphidiploids were produced via unreduced gametes for spontaneous doubling of chromosomes of triploid T. turgidum–Ae. umbellulata F1 hybrid plants. Seven pairs of U chromosomes of Ae. umbellulata were distinguished by fluorescence in situ hybridization (FISH) probes pSc119.2/(AAC)5 and pTa71. Polymorphic FISH signals were detected in three (1U, 6U and 7U) of seven U chromosomes of four Ae. umbellulata accessions. The chromosomes of the tetraploid wheat parents could be differentiated by probes pSc119.2 and pTa535, and identical FISH signals were observed among the three accessions. All the parental chromosomes of the amphidiploids could be precisely identified by probe combinations pSc119.2/pTa535 and pTa71/(AAC)5. The T. turgidum–Ae. umbellulata amphidiploids possess valuable traits for wheat improvement, such as strong tillering ability, stripe rust resistance and seed size-related traits. These materials can be used as media in gene transfers from Ae. umbellulata into wheat.

Effects of TGF-β Overexpression via rAAV Gene Transfer on the Early Repair Processes in an Osteochondral Defect Model in Minipigs

Background: Application of the chondrogenic transforming growth factor beta (TGF-β) is an attractive approach to enhance the intrinsic biological activities in damaged articular cartilage, especially when using direct gene transfer strategies based on the clinically relevant recombinant adeno-associated viral (rAAV) vectors. Purpose: To evaluate the ability of an rAAV–TGF-β construct to modulate the early repair processes in sites of focal cartilage injury in minipigs in vivo relative to control (reporter lacZ gene) vector treatment. Study Design: Controlled laboratory study. Methods: Direct administration of the candidate rAAV–human TGF-β (hTGF-β) vector was performed in osteochondral defects created in the knee joint of adult minipigs for macroscopic, histological, immunohistochemical, histomorphometric, and micro–computed tomography analyses after 4 weeks relative to control (rAAV- lacZ) gene transfer. Results: Successful overexpression of TGF-β via rAAV at this time point and in the conditions applied here triggered the cellular and metabolic activities within the lesions relative to lacZ gene transfer but, at the same time, led to a noticeable production of type I and X collagen without further buildup on the subchondral bone. Conclusion: Gene therapy via direct, local rAAV–hTGF-β injection stimulates the early reparative activities in focal cartilage lesions in vivo. Clinical Relevance: Local delivery of therapeutic (TGF-β) rAAV vectors in focal defects may provide new, off-the-shelf treatments for cartilage repair in patients in the near future.

Genetic modifications as a future prospect in the improvement of the major qualitative traits of cereals. A review

Progress in cereals transformation which can be observed for last two decades has great importance in the development of plant science and agriculture. So far, non-vector techniques, particularly direct gene transfer using „gene gun”, have been often applied in cereals transformation. However, agrobiotechnology achievements enabled cereals transformation with the soil bacterium Agrobacterium tumefaciens. Initially, it was believed that this technique cannot be applied to cereals because monocotyledones are outside the host range of the crown gall disease. Nowadays, the top five cereals with the highest economic significance – rice (Oryza sativa L.), maize (Zea mays L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) are quite efficiently transformed by A. tumefaciens. By means of molecular genetic tools it is possible to obtain cereals with new, improved traits. The present paper is focused on agricultural development which can by observed by the application of GM cereals tolerant to biotic and abiotic stress factors. Moreover, we summarized the latest achievements in cereals transformation.

GLP-1-mediated gene therapy approaches for diabetes treatment

Glucagon-like peptide (GLP)-1 is an incretin hormone with several antidiabetic functions including stimulation of glucose-dependent insulin secretion, increase in insulin gene expression and beta-cell survival. Despite the initial technical difficulties and profound inefficiency of direct gene transfer into the pancreas that seriously restricted in vivo gene transfer experiments with GLP-1, recent exploitation of various routes of gene delivery and alternative means of gene transfer has permitted the detailed assessment of the therapeutic efficacy of GLP-1 in animal models of type 2 diabetes (T2DM). As a result, many clinical benefits of GLP-1 peptide/analogues observed in clinical trials involving induction of glucose tolerance, reduction of hyperglycaemia, suppression of appetite and food intake linked to weight loss have been replicated in animal models using gene therapy. Furthermore, GLP-1-centered gene therapy not only improved insulin sensitivity, but also reduced abdominal and/or hepatic fat associated with obesity-induced T2DM with drastic alterations in adipokine profiles in treated subjects. Thus, a comprehensive assessment of recent GLP-1-mediated gene therapy approaches with detailed analysis of current hurdles and resolutions, is discussed.