Effect of biotherapic of Alternaria solani on the early blight of tomato-plant and the in vitro development of the fungus

Background: homeopathy is a means permitted in organic agriculture to control disease and plagues; biotherapics are a practical means for farmers to intervene on the health of plants in agro-ecological systems of production. Tomato-plants can be affected by several diseases, one of the most significant ones in Brazil is early blight, caused by fungus Alternaria solani, due to the damage it causes and its wide distribution in the country. Aims: to establish whether a biotherapic of A. solani may interfere on the in vitro development of the fungus and whether it affects the severity of early blight on tomato-plants in greenhouse. Methods: the effect of the biotherapic on the fungus was evaluated through the percentage of germinated spores under microscope and the growth of colonies in a culture medium. Treatments used were: biotherapic 26cH, 27cH, 28cH, 29cH and 30cH; sterilized distilled water; and diluted and agitated hydroalcoholic solution. The effect of the biotherapic on the development of disease was evaluated in 4 experiments in greenhouse. Plants were kept in vases and subjected to artificial inoculation of the fungus after the application of treatments. Evaluation of disease was carried out through diagrammatic scale. Results: no treatment affected the germination of spores or the development of fungus colonies in the culture medium. In the first test, treatment 26cH differed from water in Tukey’s test at 5% but did not differed from diluted and agitated hydroalcoholic solution. In the second test, treatments 27cH and 28cH showed significant difference from both water and hydroalcoholic solution with an average control of disease of 57% and 62% respectively. The other 2 tests did nor exhibit any significant effect. Conclusions: there was no direct effect of the biotherapic on the fungus, but there was an effect on the severity of the disease. Factors affecting the efficiency of the biotherapic must be better understood before it can be recommended to farmers for the management of early blight in tomato-plants.

The effects of supplemented sericin on in vitro maturation and preimplantation development of mouse embryos: An experimental study

Background: Mouse embryo culture condition is an essential part of transgenic, reproductive and developmental biology laboratories. Mouse embryonic culture media may have a high risk of serum contamination with pathogens. Objective: To investigate the effect of sericin as an embryo culture medium supplement on in vitro maturation (IVM), in vitro fertilization (IVF), and development of the preimplantation embryo in mice. Materials and Methods: The effects of sericin at three concentrations (subgroups) of 0.1%, 0.5%, and 1% as a medium supplement on IVM, IVF, and in vitro development of mouse embryos were separately investigated and compared with a sericin-free (control) group. The cumulative effect of the three concentrations was evaluated for IVM + in vitro development and IVF + in vitro development as follow-up groups. Results: In the IVM group, compared to the control group, the number of oocysts reaching the MII stage was significantly higher when 1% sericin was used (161/208 = 77.4%). No significant results were observed in the IVF and in vitro development groups with different concentrations of sericin compared to the control group. Among the follow-up groups, in the IVM + in vitro development group, the number of oocytes was higher after passing the IVM and IVF and reaching the blastocysts stage when 1% sericin was used, compared with other sericin subgroups. A significant difference was also noted when compared with the control group (p = 0.048). The IVF + in vitro development study group, on the other hand, did not show any significant relationship. Conclusion: It can be concluded that 1% sericin can be used as a supplement in mouse embryo cultures to improve the IVM rate. Also, based on the findings, sericin appears to be an effective supplement which can have a positive effect on the development of embryos derived from IVM. Key words: Sericin, In vitro maturation, In vitro fertilization, Preimplantation embryo, Culture medium, Mice.

Antioxidant Activity and Anti-Apoptotic Effect of the Small Molecule Procyanidin B1 in Early Mouse Embryonic Development Produced by Somatic Cell Nuclear Transfer

As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H2O2) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H2O) and oxygen (O2) in order to reduce the harm caused by H2O2. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H2O2 accumulates excessively, it produces radical-(HO-) through Fe2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H2O2 by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.

Relationship between auxins and cytokinins in the growth and organogenesis of Ocimum basilicum L. ‘Grecco a Palla’

The in vitro development of a plant is controlled by factors that promote a series of plant responses, which interfere with tissue organogenesis and morphology. For plants of the family Lamiaceae, these factors remain unknown or poorly understood, hindering in vitro cultivation of these plants. The basil cultivar ‘Grecco a palla’ has attractive chemical properties for medicinal, pharmaceutical, and cosmetic industries; however, its production is limited due to the lack of appropriate cultivation conditions. Two types of explants of this species (nodal segments and stem apexes) were grown in culture media with auxin and cytokinin, and their development was followed for 60 days. During in vitro cultivation, both explants were subjected to higher concentrations of plant growth regulators (PGRs) produced only calluses, without induction of shoots. Small amounts of regulators favored hyperhydricity as nodal segments or stem apexes in the absence of PGRs produced plants with disturbances, including brittle, light green, and thick leaves. In this case, there was an increase in the cell layers of palisade parenchyma, which had large cell spaces and larger cells. This tissue also advanced to spongy parenchyma and compressed it. The stomatal density was low; however, the stomata were larger with additions mainly in the guard cells and the stomatic opening. Therefore, stem apexes in the absence of PGRs produced more vigorous plants, whereas nodal segments with low amounts of cytokinins and auxins developed a well-branched and abundant root system.

Combinations of Trolox and ascorbic acid have a beneficial effect on in vitro maturation of pig oocytes

The poor in vitro development of pig oocytes and embryos has been blamed on oxidative stress. We sought to find out if combinations of Trolox (T), a synthetic and cell-permeable derivative of vitamin E, and ascorbic acid (AA) could improve the maturation rates of in vitro cultured pig oocytes. Pig oocytes underwent maturation for 44–45 h in medium M 199 supplemented with 0 μM T + 0 μM AA, 100 μM T + 250 μM AA, 300 μM T + 250 μM AA, 100 μM T + 750 μM AA or 300 μM T + 750 μM AA. These combinations were chosen based on previous research conducted in our laboratory and on the available literature. After maturation, several parameters were assessed: cumulus oophorus expansion, oocyte viability (based on the presence of metabolic activity versus membrane damage), extrusion of the first polar body, mitochondrial membrane potential (MMP), pronucleus formation, and embryo development after fertilization. All antioxidant combinations significantly improved cumulus expansion and formation of the first polar body. The best was 300 μM T + 250 μM AA for the first characteristic and 300 μM T + 750 μM AA for the second. Antioxidant presence in the maturation media increased the percentages of viable oocytes but not significantly. MMP was not significantly modified by the addition of antioxidant combinations. We also found that a low concentration of T (100 µM) mixed with a high concentration of AA (750 µM) in the oocyte maturation media led to significantly higher rates of both female and male pronuclei formation and also enhanced embryo development to the morula stage. Therefore, we recommend this combination to improve the in vitro maturation media of pig oocytes.

Ecological Performance of Multifunctional Pesticide Tolerant Strains of Mesorhizobium Sp. In Chickpea With Recommended Pendimethalin, Ready-mix of Pendimethalin and Imazethpyr, Carbendazim and Chlorpyrifos Application

The present study was designed to screen the Mesorhizobium strains (50) for tolerance in four recommended pesticides for chickpea. It was followed by in-vitro development of robust pesticide tolerant strains by growth in pesticides amended media over several generations. Further, verification of the multifunctional traits of pesticide tolerant mesorhizobia under pesticide stress was conducted in-vitro. Among different pesticides, significantly high tolerance in Mesorhizobium strains was observed with recommended doses of pendimethalin (37%) and ready-mix (36%) followed by chlorpyrifos (31%) and carbendazim (30%), on an overall basis. Based on multifunctional traits, Mesorhizobium strains viz. MR2, MR17 and recommended MR33 were the most promising. Ecological performance of the potential Mesorhizobium strains alone and in dual-inoculation with recommended PGP rhizobacterium strain RB-1 (Pseudomonas argenttinensis JX239745.1) was further analyzed in field following standard pesticide application in PBG-7 and GPF-2 chickpea varieties for two consecutive rabi seasons (2015 and 2016). Dual-inoculant treatments; recommended RB-1+MR33 (4.1%) and RB-1+MR2 (3.8%) significantly increased the grain yield over Mesorhizobium alone treatments viz MR33 and MR2, respectively. Grain yield in PBG7 variety was significantly affected (7.3%) by the microbial inoculant treatments over GPF2 variety. Therefore, the potential pesticide tolerant strains MR2 and MR33 can be further explored as compatible dual-inoculants with recommended RB-1 for chickpea under environmentally stressed conditions (pesticide application) at multiple locations. Our approach using robust multifunctional pesticide tolerant Mesorhizobium for bio-augmentation of chickpea with might be helpful in the formulation of effective bio-inoculants consortia in establishing successful chickpea–Mesorhizobium symbiosis.

Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.