Temporary Immersion System Improves Regeneration of in vitro Irradiated Recalcitrant Rice Embryogenic Calli

The development of gamma rays mutant rice lines would be a solution for introducing variability in already farmer using varieties. In vitro gamma (60Co) mutagenesis reduces chimeras and allows a faster selection of desired traits but requires laboratory process optimization. The objective of the present work was the in vitro establishment of a recalcitrant rice embryogenic calli, the determination of its sensitivity to gamma radiation (Co-60), sequencing MATK and Rubisco for identification purposes, as well as generation optimization. The radiosensitivity of embryogenic calli resulted in an LD50 of 110Gy, while the 20% lethal dose was 64Gy. All sequenced genes matched perfectly with already reported MATK and Rubisco O. sativa genes with a clear SNP that identifies the local variety related to the southeast Asia Region. Callus induction improves with an MS with 2mg/L 2,4D, and the regeneration was achieved with an MS medium with 3mg/L BAP and 0,5mg/L NAA. The optimized radiation condition was 60Gy with an 83% regeneration in a semisolid medium, allowing a balance between mutation and regeneration. When increased to 80Gy, the regeneration rate falls to 29%. An immersion system (RITA®) of either 60 or 120 seconds every 8hours allowed a systematic and homogeneous total regeneration of the recalcitrant line, in contrast with the semisolid medium that resulted in positive but irregular regeneration. Other well-known recalcitrant cultivars, CR1821, CR1113 also had an improving regeneration in the immersion system, demonstrating its potential use for recalcitrant materials. To our knowledge, this is the first report on using an immersion system to allow regeneration of gamma-ray mutants from recalcitrant rice materials.

Is It Possible to Produce Certified Hazelnut Plant Material in Sicily? Identification and Recovery of Nebrodi Genetic Resources, in vitro Establishment, and Innovative Sanitation Technique From Apple Mosaic Virus

Eight Sicilian cultivars of hazelnut (Corylus avellana L.), namely-Curcia, Nociara Collica, Panottara Collica, Panottara Galati Grande, Parrinara, Panottara Baratta Piccola, Enzo, and Rossa Galvagno, registered into the Italian Cultivar Register of fruit tree species in 2017 were selected from Nebrodi area and established in vitro. The aim of the work was to carry out the sanitation of the cultivars and get virus-free plants from the most important viral pathogen threat, the apple mosaic virus. Virus-free plant material is essential for the production of certified plants from Sicilian hazelnut cultivars, complying the CE (cat. CAC) quality and the technical standards established in 2017 for voluntary certification by the Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF). In this study, we investigated the possibility of establishing in vitro true-to-type and virus-free hazelnut plantlets via the encapsulation technology of apexes. The in vitro shoot proliferation rates were assessed for the different cultivars, sampling periods, temperature treatments, and type of explant used for culture initiation. Viability, regrowth, and conversion rates of both conventional meristem tip culture (MTC) and not conventional (MTC combined with the encapsulation technology) sanitation techniques were evaluated.

Evaluation of different antioxidants during in vitro establishment of allspice (Pimenta dioica L. Merrill): a recalcitrant species

Objective: To evaluate the effect of different antioxidant agents during in vitro establishment of allspice (Pimenta dioica L. Merrill). Design/methodology/approach: The effect of different antioxidant agents (Methylene blue, L-cysteine, and silver nanoparticles [AnNPs]) added to Murashige and Skoog culture medium at different concentrations were studied during axenic establishment of P. dioica. A completely randomized experimental design was used. All trials were performed in triplicate. The percentage of survival, oxidation, contamination was determined, the phenols content, antioxidant capacity and lipid peroxidation. Results: The highest survival occurred with the addition of L-cysteine. The lowest percentages oxidation were observed in explants treated with L-cysteine. Treatments with 100 and 200 mg L-1 AgNPs had the lowest contamination values. L-cysteine and 50 and 100 mg L-1 AgNPs resulted in an increase in the content of soluble phenols. The highest contents of cell wall-linked phenols were obtained in treatments with 200 mg L-1 methylene blue, L-cysteine, and 200 mg L-1 AgNPs. In this study, all treatments had a reaction of scavenging/reduction mechanisms free radicals. The highest content of malondialdehydes was observed in the control treatment and 200 mg L-1 methylene blue. The highest content of malondialdehydes was observed in the control treatment and 200 mg L-1 methylene blue. Limitations on study/implications: The highest percentage of oxidation was observed in the control treatments, 100 and 200 mg L-1 methylene blue, causing cell death. Findings/conclusions: The addition of L-cysteine to the culture medium is alternative to reduce oxidation during in vitro introduction of P. dioica.

Unraveling Factors Affecting Micropropagation of Four Persian Walnut Varieties

Walnuts are considered recalcitrant to tissue culture, with a great genetic determinism on all stages of micropropagation; while other factors, also with great impact, become more complicated with the reproduction of newly realized varieties. In this research, a holistic approach aimed to determine the influence of genotype and the nutritive formulation throughout the whole cycle of micropropagation of four Persian walnut varieties (Juglans regia L.) was presented. During the in vitro establishment it was determined that besides genotype and culture medium, the effect of collection season and the likely interaction amongst factors had a great influence on the successful introduction of all four genotypes. However, all cultures were affected by a deep decay, being necessary the introduction of ethylenediamine di-2-hydroxyphenyl acetate ferric, as iron source, and Phloroglucinol in both Murashige and Skoog (1962) and the corrected Driver and Kuniyuki (1987) formulations. These modifications allowed the stabilization of cultures, maintaining thereafter a steady quality. Either proliferation, rooting and ex vitro survival of four clones were affected by the culture medium, obtaining the best results with the corrected Driver and Kuniyuki (1987) formulation. Finally, in vitro plants produced from all clones were acclimated with high survival rates (75.9–91.1% for the best culture medium), depending of clone and the culture medium used. Microsatellite analysis showed that micropropagated plants maintained the same genetic profiles of their corresponding donor trees. These results might contribute to deepening of the understanding of factors that determine the success of micropropagation of walnuts, and the extents of its influence; whereas, it sets the basis for the commercial micropropagation of all four clones.

Cultivar and Phenological Stage Effects on the Success of In Vitro Meristem Culture and GLRaV-3 Elimination of Croatian Autochthonous Grapevine Cultivars

The population of Croatian autochthonous cultivars has a high degree of infection with economically important viruses, so it is necessary to carry out the elimination of the viruses in some cultivars to obtain healthy planting material. In this research, we tested in vitro meristem culture establishment on 18 autochthonous cultivars with different viral infections and the possibility of GLRaV-3 elimination through in vitro meristem culture. Plant material was sampled in a vineyard in two phenological stages, 10 days before flowering and 10 days after flowering of the grapevine. Apical meristem explants (1 mm) were placed into the MS culture medium supplemented with 0.5 mg/L benzyl adenine (BA) and 0.05 mg/L indol-3-acetic acid (IAA), and their survival, regeneration, and rooting were monitored. The results showed that the cultivar and the growth phase have a significant impact on the success of in vitro culture. In all cultivars studied higher success of in vitro culture establishment parameters (survival, regeneration, and rooting) was obtained in the case of explants sampled after flowering, with the exception of one cultivar for explants survival. Contrary to expectations, genotypes infected with three viruses (GLRaV-1, GLRaV-3, and GFLV) showed better results than genotypes infected with one or two viruses. The results showed successful in vitro establishment of Croatian autochthonous cultivar and GRLaV-3 elimination in one cultivar. However, due to the significant effect of cultivar, for routine application of this in vitro protocol on more than 100 autochthonous cultivars in need of sanitation, further studies should be conducted.

Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

Light sources on the germination and initial in vitro establishment of Schomburgkia crispa Lindl., a species of the Brazilian Cerrado

ABSTRACT: Light is one of the factors that influence the germination and initial establishment of orchids under in vitro cultivation. This study evaluated the effect of different light sources on these stages in in vitro cultivation of Schomburgkia crispa Lindl. After sowing in an aseptic environment, we stored the cultures in a screened greenhouse (natural light) or in a growth room with the following light sources: 3,000 K yellow LED; 6,500 K white LED [1]; 6,500 K white LED [2]; or 6,500 K white fluorescent lamp (control). We assessed germination percentage and initial seedling establishment at 45 and 90 days after sowing. Light did not influence the germination of S. crispa. However, the use of 3,000 K LED provided a faster initial establishment of S. crispa when compared to the other light sources, also presenting lower seedling mortality. Thus, the light source 3,000 K LED is a potential substitute for the 6,500 K fluorescent lamps and LEDs used in growth rooms in in vitro culture laboratories.