Bruno Delbreil

The flowering locus Hr colocalizes with a major QTL affecting winter frost tolerance in Pisum sativum L

An understanding of the genetic determinism of frost tolerance is a prerequisite for the development of frost tolerant cultivars for cold northern areas. In legumes, it is not known to which extent vernalization requirement or photoperiod responsiveness are necessary for the development of frost tolerance. In pea (Pisum sativum L.) however, the flowering locus Hr is suspected to influence winter frost tolerance by delaying floral initiation until after the main winter freezing periods have passed. The objective of this study was to dissect the genetic determinism of frost tolerance in pea by QTL analysis and to assess the genetic linkage between winter frost tolerance and the Hr locus. A population of 164 recombinant inbred lines (RILs), derived from the cross Champagne x Terese was evaluated both in the greenhouse and in field conditions to characterize the photoperiod response from which the allele at the Hr locus was inferred. In addition, the population was also assessed for winter frost tolerance in 11 field conditions. Six QTL were detected, among which three were consistent among the different experimental conditions, confirming an oligogenic determinism of frost tolerance in pea. The Hr locus was found to be the peak marker for the highest explanatory QTL of this study. This result supports the hypothesis of the prominent part played by the photoperiod responsiveness in the determinism of frost tolerance for this species. The consistency of three QTL makes these positions interesting targets for marker-assisted selection.

Abiotic Stress Responses in Legumes: Strategies Used to Cope with Environmental Challenges

Legumes are well recognized for their nutritional and health benefits as well as for their impact in the sustainability of agricultural systems. The threatening scenario imposed by climate change highlights the need for concerted research approaches in order to develop crops that are able to cope with environmental stresses, while increasing yield and quality. During the last decade, some physiological components and molecular players underlying abiotic stress responses of a broad range of legume species have been elucidated. Plant physiology approaches provided general outlines of plant responses, identifying stress tolerance-related traits or elite cultivars. A thorough identification of candidate genes and quantitative trait loci (QTLs) associated with these traits followed. Model legumes like Medicago truncatula, Lotus japonicus, and more recently, Glycine max provided valuable translational approaches for dissecting legume responses to abiotic stresses. The challenge now focuses on the translation of the information gained in model systems in controlled environments to crops grown under field conditions. In this review, we provide a general overview of the recent achievements on the study of abiotic stress responses in a broad range of model, grain and forage legumes species, highlighting the different approaches used. Major accomplishments, as well as limitations or drawbacks are discussed across the different sections. Some perspectives regarding new approaches for screening, breeding or engineering legumes with desirable abiotic stress resistance traits are anticipated. These advances will support the development of legumes better adapted to environmental constraints, tackling current demands on modern agriculture and food production presently exacerbated by global climate changes.

A proteomic approach to decipher chilling response from cold acclimation in pea (Pisum sativum L.).

Two pea lines (Pisum sativum L.) with contrasted behaviours towards chilling and subsequent frost were studied by a proteomic approach to better understand cold acclimation. Following a chilling period, the Champagne line becomes tolerant to frost whereas Terese remains sensitive. Variance analysis allowed to select 260 statistically variable spots with 68 identified proteins (35 in leaves, 18 in stems, and 15 in roots). These proteins were shared out in proteins related to chilling response or cold acclimation. The better adaptation of Champagne to chilling might be related to a higher content in proteins involved in photosynthesis and in defence mechanisms. Moreover Champagne might prevent freezing damage particularly thanks to a higher constitutive expression of housekeeping proteins related to Terese. After three days of subsequent frost, proteomes of previously chilled plants also showed significant differences compared to unchilled plants. Out of 112 statistically variable spots (44 in leaves, 38 in stems, and 30 in roots), 32 proteins were identified. These proteins were related to frost response or frost resistance. It seems that Champagne could resist to frost with the reorientation of the energy metabolism.

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

SummaryTwenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.

Association of sugar content QTL and PQL with physiological traits relevant to frost damage resistance in pea under field and controlled conditions

To increase yield in pea (Pisum sativum L.), autumn sowing would be preferable. Hence, frost tolerance of pea became a major trait of interest for breeders. In order to better understand the cold acclimation in pea, Champagne a frost tolerant line and Terese, a frost sensitive line, and their recombinant inbred lines (RIL) were studied. RIL frost tolerance was evaluated by a frost damage scale under field as well as controlled conditions. A quantitative trait loci (QTL) approach was used to identify chromosomal regions linked to frost tolerance. The detected QTL explained from 6.5 to 46.5% of the phenotypic variance. Amongst them, those located on linkage groups 5 and 6 were consistent with over all experiments, in field as well as in controlled environments. In order to improve the understanding of the frost tolerance mechanisms, several cold acclimation key characters such as concentration of sugars, electrolyte leakage, osmotic pressure, and activity of RuBisCO were assessed. Some of these physiological QTL colocalised with QTL for frost damage, in particular two raffinose QTL on LG5 and LG6 and one RuBisCO activity QTL on LG6, explaining 8.8 to 27.0% of the phenotypic variance. In addition, protein quantitative loci were mapped; some of them colocalised with frost damage and physiological QTL on LG5 and LG6, explaining 16.0–43.6% of the phenotypic variance. Raffinose metabolism and RuBisCO activity and its effect on photosynthesis might play a major role in cold acclimation of pea.

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

SummarySomatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.

Exploring chloroplastic changes related to chilling and freezing tolerance during cold acclimation of pea (Pisum sativum L.).

Pea (Pisum sativum L.) productivity is linked to its ability to cope with abiotic stresses such as low temperatures during fall and winter. In this study, we investigate the chloroplast-related changes occurring during pea cold acclimation, in order to further lead to genetic improvement of its field performance. Champagne and Térèse, two pea lines with different acclimation capabilities, were studied by physiological measurements, sub-cellular fractionation followed by relative protein quantification and two-dimensional DIGE. The chilling tolerance might be related to an increase in protein related to soluble sugar synthesis, antioxidant potential, regulation of mRNA transcription and translation through the chloroplast. Freezing tolerance, only observed in Champagne, seems to rely on a higher inherent photosynthetic potential at the beginning of the cold exposure, combined with an early ability to start metabolic processes aimed at maintaining the photosynthetic capacity, optimizing the stoichiometry of the photosystems and inducing dynamic changes in carbohydrate and protein synthesis and/or turnover.

A high-density genetic map of the Medicago truncatula major freezing tolerance QTL on chromosome 6 reveals colinearity with a QTL related to freezing damage on Pisum sativum linkage group VI

Freezing is one of the most serious abiotic stress factors that affect cool-season legumes. It limits species geographic distribution and causes severe yield losses. Improving tolerance to freezing has long been a main concern for legume breeders. Medicago truncatula Gaertn. has been selected as a model species for legume biology. Various studies have shown significant macrosynteny between M. truncatula and agronomically important crop legumes. A major freezing tolerance quantitative trait locus (QTL), herein referred to as Mt-FTQTL6, was previously identified on M. truncatula chromosome 6. The physical location of this QTL was determined in this study and its corresponding chromosomal interval was enriched with additional markers. Markers were first developed using the draft sequence of M. truncatula euchromatin (release versions Mt3.0 and Mt3.5). Because Mt-FTQTL6 was found to coincide with an assembly gap, the Glycine max (L.) Merr. genome sequence was also used to generate markers. Five Mt-FTQTL6-linked markers were found to be common to a region on Pisum sativum L. linkage group VI harboring a QTL for freezing damage. A subset of markers was tested for transferability across 11 additional legume species. This study lays the groundwork for identifying the molecular basis of Mt-FTQTL6. Cross-legume markers will be useful in future efforts aiming to investigate the conservation of Mt-FTQTL6 in cool-season legumes and subsequently the existence of common mechanisms for response to freezing between M. truncatula and crop legumes.

Isolation and Characterization of Long-term Embryogenic Lines in Asparagus officinalis L.

Summary Somatic embryogenesis was obtained from 14 Asparagus officinalis L. genotypes by in vitro culture of different explant types such as cladophylls, apices and isolated mesophyll cells. Explants first induced on a culture medium containing growth regulators produced calli and developed few somatic embryos when transferred on a hormone-free medium. During this development phase, we isolated a new type of friable tissues that produced abundant somatic embryos. The frequencies of emergence of these highly embryogenic tissues were estimated between 10 −5 and 10 −6 . These tissue were habituated; being established as long-term embryogenic lines by repeated subcultures on hormone-free medium, continuously they produced numerous somatic embryos, some of them converting to whole plants. Histological data showed these long-term embryogenic lines to grow by recurrent embryogenesis arising, from single epidermal cells of preexisting embryos.

Transcriptome analysis in pea allows to distinguish chilling and acclimation mechanisms

In order to distinguish chilling and freezing tolerance mechanisms in pea, responses to cold exposure were compared between the freezing tolerant line Champagne and the sensitive line Terese. Global gene expression was considered in the two lines and associated with morphological, histological and biochemical approaches. The chilling tolerance in both lines was related to responses of the CBF, COR and LEA genes belonging to the CBF regulon, with greater earliness of expression in the Champagne genotype. The freezing tolerance, only observed in Champagne, was associated with acclimation processes such as cellular osmotic stabilization, photosynthesis modifications, antioxidants production, modifications in hormone metabolism, cell wall composition and dynamics.