Simple solution to preserve plant samples for microbiome analyses.

Culture-independent survey techniques are fundamental tools when assessing plant microbiomes. These methods rely on DNA that is carefully preserved after collecting samples to achieve meaningful results. Immediately freezing samples to -80°C after collection is considered one of the most robust methods for preserving samples before DNA extraction but is often impractical. Preservation solutions can solve this problem, but commercially available products are expensive, and there is limited data comparing their efficacy with other preservation methods. In this study, we compared the impact of three methods of sample preservation on plant microbiome surveys: (1) RNAlater, a proprietary preservative, (2) a home-made salt-saturated dimethyl sulphoxide preservation solution (DESS), and (3) freezing at -80°C. DESS-preserved samples, stored at room temperature for up to four weeks, did not show any significant differences to samples frozen at -80°C, while RNAlater inflated bacterial alpha diversity. Preservation treatments did not distinctively influence fungal alpha diversity. Our results demonstrate that DESS is a versatile and inexpensive preservative of DNA in plant material for diversity analyses of fungi and bacteria.

Biolistics-Mediated Gene Delivery in Sugarcane.

Biolistic transformation is one of two popular methods for introducing genes into sugarcane. However, unlike Agrobacterium-mediated transformation, the efficiency of gene transfer into sugarcane cells, using the biolistic method is very high. In addition to this, the biolistic transformation method is independent of the explant genotype or tissue. It also has the advantage that a minimum DNA sequence of linearized plasmid can be used, thus eliminating the introduction of undesirable plasmid derived genes, delivering low-copy transgenic events. In this chapter, we describe the method for efficient delivery of genes into sugarcane cells using a biolistic approach.

Overexpression of TaNAC69 leads to enhanced transcript levels of stress up-regulated genes and dehydration tolerance in bread wheat.

NAC proteins are plant-specific transcription factors and enriched with members involved in plant response to drought stress. In this study, we analyzed the expression profiles of TaNAC69 in bread wheat using Affymetrix Wheat Genome Array datasets and quantitative RT-PCR. TaNAC69 expression was positively associated with wheat responses to both abiotic and biotic stresses and was closely correlated with a number of stress up-regulated genes. The functional analyses of TaNAC69 in transgenic wheat showed that TaNAC69 driven by a barley drought-inducible HvDhn4s promoter led to marked drought-inducible overexpression of TaNAC69 in the leaves and roots of transgenic lines. The HvDhn4s:TaNAC69 transgenic lines produced more shoot biomass under combined mild salt stress and water-limitation conditions, had longer root and more root biomass under polyethylene glycol-induced dehydration. Analysis of transgenic lines with constitutive overexpression of TaNAC69 showed the enhanced expression levels of several stress up-regulated genes. DNA-binding assays revealed that TaNAC69 and its rice homolog (ONAC131) were capable of binding to the promoter elements of three rice genes (chitinase, ZIM, and glyoxalase I) and an Arabidopsis glyoxalase I family gene, which are homologs of TaNAC69 up-regulated stress genes. These data suggest that TaNAC69 is involved in regulating stress up-regulated genes and wheat adaptation to drought stress.