Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

Chemical fertilizers are used only in a limited way under dry farming conditions in India because of their high cost and the risk of crop failures due to drought Several alternative strategies are being evaluated to reduce the cost of supplying N to castor bean without adversely affecting yield. We report the results of a two-year study on the possible replacement of fertilizer nitrogen by green leaves from Leucaena leucocephala for production of castor beans on a low fertility Alfisol Bean yield and dry matter production were significantly higher when 50 kg N/ha was applied as green leaves compared with 50 kg N/ha as urea. Among substitution levels, 37.5 kg N/ha as green leaves at planting followed by 12.5 kg N/ha as urea at 45 days after planting was found to be the most effective combination. Across all the substitution levels, addition of nitrogen as green leaves at planting was found to be better than application at 45 days after planting. Nutrient and water use efficiencies were higher when the N was applied as green leaves compared to urea. Nitrogen use efficiency was higher for green leaves than for urea under limited rainfall conditions. The overall data indicated that 75 percent of the fertilizer N requirement of castor bean can be supplied by leaves of Leucaena leucocephala when applied at planting.