MicroRNAs (miRNAs) play critical roles in the development of animals and plants. Characterizing the stage- and tissue-specific expression of miRNAs that potentially regulate target transcription factor expression is becoming more important for understanding the regulatory mechanisms of critical events during plant development. A simple method for purifying small RNAs from seeds is described, as well as an efficient non-radioactive labelling system for making miRNA probes. In Arabidopsis thaliana seed extracts, low molecular-weight (LMW) RNAs (e.g. 5S rRNA, tRNA and miRNA) were separated from high molecular-weight (HMW) nucleic acids (e.g. 28S and 18S rRNA, mRNA and genomic DNA) by fractionation using isopropanol. HMW RNAs precipitated in 20% isopropanol, while most LMW RNAs remained in the supernatant. The purified LMW RNAs were used successfully for RNA gel blotting to detect miRNAs expressed in Arabidopsis and tomato (Lycopersicon esculentum) seeds. To increase the detection sensitivity of the microRNA probes, additional digoxigenin-labelled uridine triphosphates (UTPs) were incorporated into the miRNA probes by designing template oligo DNAs with three extra adenines (A) at each end of their sequence. These DNA oligomers were used to make double-stranded DNA templates for miRNA probe synthesis. This probe (termed AAAPLUS) exhibited stronger signals than normal probes. A technique was also developed to quickly screen expressed miRNAs in seeds using a miniblot system, which enabled simultaneous examination with multiple miRNA probes. This method provides a simple alternative to microRNA microarrays to identify the major miRNAs expressed in seeds.

The enhanced laboratory and field emergence characteristics of osmo- and halo-primed tomato seeds (cv. Pusa Ruby) were related to changes in hydration–dehydration kinetics, modified sorption properties and nuclear magnetic resonance (NMR) relaxation behaviour of humidified and imbibed seeds. Water sorption isotherms were constructed for primed and unprimed seeds by equilibrating to different water activities (aw) at 25°C. Analysis of the isotherms by the D'Arcy–Watt equation revealed that priming reduced the number of strong binding sites and the associated water content, and increased significantly the number of weak binding sites and the associated water content. This redistribution of water, which increased the availability of seed water, may be the reason for the higher speed of germination of primed seeds. The changes in transverse relaxation time (T2) of seed water and its components, measured in vivo using nuclear magnetic resonance spectroscopy, showed interesting differences between primed and unprimed seeds. With an increase in humidification time, the T2 of primed seeds could be resolved into three components with varying mobilities, while the control seeds had only two components until 10 d of humidification. During imbibition, the third component appeared after 2 and 6 h in primed and control seeds, respectively. This component disappeared after the germination process started in all treatments. The third fraction, with very low molecular mobility, which accounted for about 40% of the proton population, was assigned to hydration water of macromolecules. Hence, we propose that better performance of primed seeds may be attributed to the modifications of seed water-binding properties and reorganization of seed water during imbibition, so as to increase the macromolecular hydration water required for various metabolic activities related to the germination process.

The involvement of ethylene in determining the time to radicle protrusion was investigated in ethylene-insensitive gain-of-function (GOF) receptor mutants in tomato and Arabidopsis, as well as in single and double loss-of-function (LOF) receptor mutants in Arabidopsis. Because ethylene evolution from seeds is coincident with radicle protrusion, and the ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene is diagnostic for seed vigour, it was hypothesized that ethylene-insensitive mutants would require more time to complete germination compared to wild-type seeds. Mutant Never Ripe (Nr) tomato seeds from two genetic backgrounds refuted this hypothesis, while experiments with wild-type seeds, treated with the ethylene action inhibitors, 2,5-norbornadiene or silver thiosulphate, supported it. However, reciprocal crosses between wild-type and Nr demonstrated that ethylene insensitivity during seed development determined subsequent time to complete germination, rather than the ability of the embryo/endosperm to perceive ethylene in the mature seed during germination. Additionally, seed quality, determined by standard vigour tests, was reduced in Nr compared to wild-type seeds, establishing a disconnection between rapid completion of germination and seed vigour. In Arabidopsis, all ethylene-insensitive GOF, and five of six single LOF mutants, required more time to complete 50% radicle protrusion, while double LOF mutants required the same, or less, time to complete germination compared to wild-type seeds. These findings support a role for ethylene perception in determining the length of time Arabidopsis seeds remain in the lag phase prior to radicle protrusion.