Dreyer Winkler (rubhat36)
The study of fungal, bacterial, and other endophytic microorganisms using high throughput DNA sequencing requires sampling and processing of plant material that eliminates phylloplane microorganisms and retains those inside the plant compartment. Leaves, stems, roots, and other plant tissues are removed from the plant, washed, surface sterilized, and stored for downstream applications. Especially in ecological studies, field work for sample collection may take place in remote locations where laboratory equipment and resources are rudimentary, and accessing samples from target plants can be challenging. This chapter serves as a guide to basic protocols in the design and sample collection for studies focused on the endophytes of leaf, stem, and root tissues.The vasculature of plants is typically colonized by a wide-range of bacteria with diverse functions. These bacteria can be sampled by pooling plant biopsies in water and then concentrating cells by centrifugation. When the extracted bacteria are added as a template for the polymerase chain reaction (PCR), sufficient DNA is generally liberated to facilitate the identification of specific taxa and characterization of bacterial community structure. The sampling technique facilitates surveys of multiple plants comprising a single crop, allowing for a more comprehensive understanding of the crop microbiome than what can be achieved when examining single plants. This technique is rapid and cost-effective, and will help researchers monitor microbes associated with vascular tissues at various stages of crop development.The microbiome is known to influence plant fitness and differs significantly between plant compartments. To characterize the communities associated with different plant compartments, it is necessary to separate plant tissues in a manner that is suitable for microbiome analysis. Here, we describe a standardized protocol for sampling the microbiomes associated with bulk soil, the apical and basal ectorhizosphere, the apical and ectorhizosphere, the rhizome, pseudostem, and leaves of Musa spp. The approach can easily be modified for work with other plants.Recent studies indicate that seed microbiomes affect germination and plant performance. However, the interplay between seed microbiota and plant health is still poorly understood. To get a complete picture of the system, a comprehensive analysis is required, comprising culture-dependent and culture-independent techniques. In this chapter, we provide a combination of methods that are established and optimized for the analysis of the seed microbiome. These include methods to (1) activate and cultivate dormant seed microbiota, (2) analyze microbiota in germinated seeds (with and without substrate), (3) quantify microbial DNA via real-time PCR, (4) deplete host DNA for amplicon and metagenome analysis, and (5) visualize seed endophytes in microtomed sections using fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). A deep understanding of the seed microbiome and its functions can help in developing new seed treatments and breeding strategies for sustainable agriculture. Delta-opioid receptor (DOR)-mediated modulation of hippocampal neural networks is involved in emotions, cognition, and in pathophysiology and treatment of mood disorders. In this study, we examined the effects of DOR agonist (SNC80) and antagonist (naltrindole) on the excitability of individual hippocampal neurons. Primary neuronal cultures were prepared from hippocampi of newborn rats and cultivated in vitro for 8-14days (DIV8-14). The effects of SNC80 naltrindole on evoked and spontaneous action potentials (APs) were measured at DIV8-9 and DIV13-14, respectively. SNC80 (100µM) potentiated spontaneous AP firing and stimulated sodium current; naltrindole had opposite effects. The stimulatory effect of 100µM of SNC80 was revoked by pre-administration of 1µM of naltrindole. SNC80 and naltrindol
