Search for: All records

Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7–9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.

Winter wheat (Triticum aestivumL.) is essential to maintain food security for a large proportion of the world’s population. With increased risk from abiotic stresses due to climate variability, it is imperative to understand and minimize the negative impact of these stressors, including high night temperature (HNT). Both globally and at regional scales, a differential rate of increase in day and night temperature is observed, wherein night temperatures are increasing at a higher pace and the trend is projected to continue into the future. Previous studies using controlled environment facilities and small field-based removable chambers have shown that post-anthesis HNT stress can induce a significant reduction in wheat grain yield. A prototype was previously developed by utilizing field-based tents allowing for simultaneous phenotyping of popular winter wheat varieties from US Midwest and advanced breeding lines. Hence, the objectives of the study were to (i) design and build a new field-based infrastructure and test and validate the uniformity of HNT stress application on a scaled-up version of the prototype (ii) improve and develop a more sophisticated cyber-physical system to sense and impose post-anthesis HNT stress uniformly through physiological maturity within the scaled-up tents; and (iii) determine the impact of HNT stress during grain filling on the agronomic and grain quality parameters including starch and protein concentration. The system imposed a consistent post-anthesis HNT stress of + 3.8 °C until maturity and maintained uniform distribution of stress which was confirmed by (i) 0.23 °C temperature differential between an array of sensors within the tents and (ii) statistically similar performance of a common check replicated multiple times in each tent. On average, a reduction in grain-filling duration by 3.33 days, kernel weight by 1.25% per °C, grain number by 2.36% per °C and yield by 3.58% per °C increase in night temperature was documented. HNT stress induced a significant reduction in starch concentration indicating disturbed carbon balance. The pilot field-based facility integrated with a robust cyber-physical system provides a timely breakthrough for evaluating HNT stress impact on large diversity panels to enhance HNT stress tolerance across field crops. The flexibility of the cyber-physical system and movement capabilities of the field-based infrastructure allows this methodology to be adaptable to different crops.

Unraveling the metabolic and phytohormonal changes in anthers exposed to heat stress would help identify mechanisms regulating heat stress tolerance during the sensitive reproductive stage. Two spring wheat genotypes contrasting for heat tolerance were exposed to heat stress during heading in controlled environment chambers. Anthers were collected from main and primary spikes for metabolic and phytohormonal profiling. A significant reduction in seed set (38%), grain number (54%) and grain weight (52%) per plant was recorded in the sensitive (KSG1177) but not in the tolerant (KSG1214) genotype under heat stress compared to control. Anther metabolite accumulation did not vary quantitatively between main and primary spikes. Hierarchical clustering of the genotypes and treatments using metabolites and phytohormones revealed a distinct cluster for KSG1177 under heat stress from that of control and KSG1214. A significant increase in N‐based amino acids, ABA, IAA‐conjugate and a decrease in polyamines and organic acids were observed in wheat anthers exposed to heat stress. Unlike KSG1214, a significantly higher accumulation of amino acids, ABA and IAA‐conjugate in anthers of the sensitive KSG1177 was recorded under heat stress. These findings provide the rationale and direction towards developing molecular markers for enhancing heat stress tolerance in wheat.