Flow cytometry distinction between Lathyrus species, landraces and in vitro regenerants

National audienc

<em>In vitro</em> biotechnology approaches now available for ‘beautiful’ vavilovia (<em>Vavilovia formosa</em>)

BAP Pôle GEAPSIInternational audienceEfficient in vitro propagation of Vavilovia formosa, plant regeneration from callus, protoplast isolation and culture to differentiated callus of V.formosa were developed and its relative nuclear DNA content by flow cytometry was established. The summation of biotechnology tools now available should foster evolutionary studies on the tribe Fabeae and, intime, V.formosa could be come a potential source of novel agronomic adaptive traits

Flow cytometry distinction between species and between landraces within Lathyrus species and assessment of true-to-typeness of in vitro regenerants

International audienceThe genus Lathyrus includes a number of neglected wild relatives of pea with potential as genetic resources for acquisition of stress resistance traits, but, due to little breeding, genotypes under culture are mainly landraces and seldom true varieties. Development of in vitro approaches for Lathyrus is also limited, and assessments of nuclear DNA content, for taxonomical or breeding purposes, are sparse. Genome size and AT/GC ratio were determined by flow cytometry, allowing for distinction between protein and forage L. sativus, L. cicera, L. ochrus and L. clymenum and the ornamental sweet pea (L. odoratus), and also between landraces within L. sativus L. and L. cicera L. In addition, explants from in vitro seedlings of eight genotypes from the five Lathyrus species above were cultivated in vitro, plant regeneration was achieved for all landraces and species, and the nuclear DNA content of the regenerants was compared with that of their mother plants, whereby the true-to-typeness of such regenerants was confirmed

In vitro production of sweet peas (Lathyrus odoratus L.) via axillary shoots

International audienceThe genus Lathyrus is best known because it includes a number of wild relatives of the protein pea which, despite being generally neglected and under-utilised, hold considerable potential as a useful genetic resource for the acquisition of interesting stress resistant traits important for a sustainable agriculture. However, also included in this genus are important commercially produced species with a significant ornamental value, among which the sweet pea (Lathyrus odoratus L.). Surprisingly though, there are no formal reports on the in vitro propagation of this species and, generally, these are scanty for in vitro approaches with all species of Lathyrus. Here, we describe simple, yet reliable strategies for the culture and multiplication of several landraces and species of Lathyrus including sweet peas

Cell wall properties and embryo development : a case study with legumes and models systems

National audienc

Flow cytometry measurements contribute to Pisum taxonomy

Pea (Pisum sativum L.) has been widely used in early hybridization studies, as model for experimental morphology and physiology, and was Mendel’s model species to untangle the laws of inheritance, which puts it at the foundation of modern genetics. Its large genome size (4775 Mbp as assessed by Feulgen method in 1976) slowed down progress in pea genomics compared with other plant species, but the recent availability of genome sequences of various legume species now permits genome wide comparison and allows to identify genes underlying agronomically important traits by combining candidate gene and synteny approaches. The efficient use of existing genomic resources is a key to success in these goals and several types of molecular marker sets as well as both transcriptome and proteome datasets exist. Despite this impressive background, and the fact that P. sativum is one of the most frequently used internal standards for flow cytometry studies with other species, there is still a need to further clarify the taxonomy within species of the genus Pisum. Thus, we have analysed by flow cytometry 42 accessions from a range of geographic origins and belonging to two wild species: P. sativum subsp. elatius (e.g. including former P. elatius and P. humile/syriacum), P. fulvum and cultivated: P. abyssinicum, P. sativum as well as some primitive P. sativum cultigens (such as subsp. transcaucassicum, asiaticum), where some of them had been identified differently or tentatively in the past based on botanical characteristics. In these studies, all materials were analysed simultaneously with Medicago truncatula as the internal standard, and with various fluorochromes (DAPI, Propidium Iodide, Chromomycine A3) to assess the relative nuclear DNA content, genome size and AT/GC ratio. For some of the species studied this is the first report on these traits

Cell wall properties and embryo development : a case study with legumes and models systems

National audienc

Flow cytometry measurements contribute to<em> Pisum taxonomy</em>

International audiencePea (Pisum sativum L.) has been widely used in early hybridization studies, as model for experimental morphology and physiology, and was Mendel’s model species to untangle the laws of inheritance, which puts it at the foundation of modern genetics. Its large genome size (4775 Mbp as assessed by Feulgen method in 1976) slowed down progress in pea genomics compared with other plant species, but the recent availability of genome sequences of various legume species now permits genome wide comparison and allows to identify genes underlying agronomically important traits by combining candidate gene and synteny approaches. The efficient use of existing genomic resources is a key to success in these goals and several types of molecular marker sets as well as both transcriptome and proteome datasets exist. Despite this impressive background, and the fact that P. sativum is one of the most frequently used internal standards for flow cytometry studies with other species, there is still a need to further clarify the taxonomy within species of the genus Pisum. Thus, we have analysed by flow cytometry 42 accessions from a range of geographic origins and belonging to two wild species: P. sativum subsp. elatius (e.g. including former P. elatius and P. humile/syriacum), P. fulvum and cultivated: P. abyssinicum, P. sativum as well as some primitive P. sativum cultigens (such as subsp. transcaucassicum, asiaticum), where some of them had been identified differently or tentatively in the past based on botanical characteristics. In these studies, all materials were analysed simultaneously with Medicago truncatula as the internal standard, and with various fluorochromes (DAPI, Propidium Iodide, Chromomycine A3) to assess the relative nuclear DNA content, genome size and AT/GC ratio. For some of the species studied this is the first report on these traits

In vitro morphogenesis from cotyledon and epicotyl explants and flow cytometry distinction between landraces of Bambara groundnut [Vigna subterranea (L.) Verdc], an under-utilised grain legume

International audienceThe morphogenetic competence of Bambara groundnut was assessed for different landraces, explant sources and media compositions. With cotyledon explants, the best callusing occurred on a medium containing 3 mg/l BAP + 0.5 mg/l NAA, while roots were produced with 3–5 mg/l BAP + 0.5 mg/l NAA. Shoots regenerated (∼6%) from cotyledons on media with BAP alone (3–5 mg/l) or combined with 0.01–0.1 mg/l NAA. Flowers were regenerated on 5 mg/l BAP + 0.5 mg/l NAA, without any intervening callus phase. With epicotyls, the highest callusing was on 3 mg/l BAP + 0.5 mg/l NAA, and shoots regenerated (15–20%) on 3 mg/l BAP alone or with NAA at concentrations that depended on the landrace studied. Regenerated shoots rooted on hormone-free medium, and plants transferred to the greenhouse were all morphologically normal and fertile. Flow cytometry showed that most regenerants were diploid and in addition permitted to distinguish between landraces according to their relative nuclear DNA content. This is the first report on de novo regeneration in vitro of Bambara groundnut, an important yet neglected legume crop

In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species <em>Medicago truncatula</em>

International audienceThe role of auxins in the morphogenesis of immature seeds of Medicago truncatula was studied, focusing on the transition from the embryo cell division phase to seed maturation. We analyzed seed development in vitro, by flow cytometry, and through the determination of the kinetics of seed fresh weight and size. Thus, seeds were harvested at 8, 10 and 12 days after pollination and cultured in vitro on a medium either without auxin or supplemented with indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) at 1 mg l(-1). All parameters studied were determined every 2 days from the start of in vitro culture. The results showed that both auxins increased the weight and size of seeds with NAA having a stronger effect than IBA. We further demonstrated that the auxin treatments modulate the transition between mitotic cycles and endocycles in M. truncatula developing seed by favoring sustained cell divisions while simultaneously prolonging endoreduplication, which is known to be the cytogenetical imprint of the transition from the cell division phase to the storage protein accumulation phase during seed development