BackgroundTILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin.ResultsHere, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified.ConclusionsThis new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

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Certified seed producers systematically select and propagate registered varieties year after year in order to maintain their uniformity and the original registered cultivar traits. However, natural mutations, spontaneous breeding between varieties and alien grain contamination can introduce undesirable variability. NRVC 980385 is a temperate japonica rice cultivar (Oryza sativa ssp. japonica) first registered in Spain in 2002. In 2005 certification tests detected a plot differing from the original traits in terms of uniformity and height suggesting the presence of a certain heterozygosis. This material was therefore seen as an opportunity to obtain newly stabilized doubled haploid (DH) lines which could compete in the Spanish short grain seed market. In this study, an in vitro anther culture protocol is defined which also covers the field tests selection to obtain four new, improved and stabilized DH derived lines ready to be registered for commercial proposes. This took just four years from the initial anther collection until new lines were grown in large scale field trials. Consequently, this protocol reduces the time for obtaining field assessed DH lines thereby having considerable advantages over other techniques by both maintaining the original registered cultivars and/or generating new derived varieties.

Carnation plantlets (Dianthus caryophyllus L.) cultured in vitro often develop morphological and physiological anomalies, a phenomenon called hyperhydricity, which impairs their survival ex vitro. When the agar concentration of the growth medium was increased (from 0 to 12 g dm -3 ), thereby reducing water availability, the hyperhydricity of those adventitious shoots regenerated from carnation petals decreased. This was accompanied by a progressive fall in the water content of shoots (94.9 to 91.4 %), fresh mass (from 57.2 to 1.8 mg), number of leaf parenchyma cell layers (from 9.3 to 7.7), and the size of these cells (from 968 to 254 µm 2 ). However, the number of regenerated shoots also decreased (17.7 in 2 g dm -3 agar to 4.3 in 12 g dm -3 ). Similarly, in ventilated tubes, which exhibit a lower relative humidity than tightly closed tubes, shoot organogenesis diminished up to 28 %, in tandem with shoot water content. Thus, relative humidity and water availability in culture vessels do not only influence shoot hyperhydricity in carnations, but also greatly affect adventitious shoot organogenesis.Additional key words: Dianthus caryophyllus, in vitro culture, leaf parenchyma.