Unravelling wild carrot differentiation in Europe: preliminary data on a candidate gene approach

Carrot is an outcrossing species and levels of gene flow between populations, and even between wild and domesticated relatives, are expected to be high. Cases of natural hybridization and introgression of crops and wild relatives have been reported. Have these events diluted any putative habitat-adapted genotypes? In other words, can we still find a correlation between wild carrot genotypes and regional/local environment? We have chosen to start addressing this question using a member of the alternative oxidase (AOX) gene family. AOX genes seem to be linked to all kinds of abiotic and biotic stress reactions. Wild carrots were sampled in an environmental gradient across Western Europe. This gradient included sampling points with more deviating conditions, such as Sierra de Guadarrama or the central Pyrenees and the French Massif Central. Phylogenetic reconstruction on this molecular marker is to be combined with geographic, climatic, and ecological evidence. So far, the preliminary results suggest the existence of a biogeographical barrier at the Pyrenees, and higher gene diversity than initially expected. From an applied point of view, diversity of functional traits is much more relevant than species diversity. Gene transfer from wild to cultivated plants has contributed to the evolution of crop species. Providing that deterioration of genetic resources and biodiversity loss have not been drastic, gene transfer from wild plants has the potential to further contribute to a (targeted) improvement of cultivars.info:eu-repo/semantics/publishedVersio

Micropropagation of a recalcitrant pine (Pinus pinea L.): An overview of the effects of ectomycorrhizal inoculation

Stone pine (Pinus pinea L.) is an economically important forest species in some regions of Iberian Peninsula. Portugal and Spain have nearly 500,000 ha of stone pine stands, representing 85% of worldwide distribution. The main use of this species is for the production of seeds (pinion) for food industry. In addition to its enormous profitability as a producer of seeds, it has beneficial impact on soil protection, dunes fixation and is a pioneer species particularly for cork and holm oaks degraded ecosystems. Stone pine plantations are today a major source of income for forestry holdings. Investments have targeted breeding, reforestation, forest management and harvesting. The maternal inheritance of desirable characteristics such as cone weight, number of seeds per cone and seed length is considerably high in this species thus encouraging the selection of seeds from “plus” trees. The selected trees have been propagated by grafting and micropropagation. However, grafting generates high variability due to scion-rootstock interaction that varies production levels. The production of clonal plants from selected seeds by micropropagation techniques has advanced very slowly due to the recalcitrance of this species in tissue culture and particularly to adventitious rooting of microshoots. Due to the tremendous importance of developing a reproducible tissue culture method for clonal propagation, a study has been carried out for over a decade to enhance rooting and acclimation. During this period of time, continuous increments in the multiplication rate and rooting frequency were achieved by introducing variations in culture media composition and conditions. Auxins, carbohydrates, light quality and duration, temperature at different concentrations and levels as well as compounds such as coumarin; salicylic acid, polyamines, etc. were tested for induction and expression phases of adventitious rooting. Despite these efforts, microshoots regenerated through organogenesis from mature embryo cotyledons failed to root or to have sustained root growth. At this point, an in vitro co-culture technique of stone pine microshoots with ectomycorrhizal-fungi was introduced to overcome the adventitious root growth cessation in vitro and improve root development during acclimation phase. An overview of the results showing the positive effect of fungal inoculation in promoting root growth in vitro and on plantlet survival during acclimation will be presented. Preliminary results of biochemical signals between Pinus pinea/Pisolithus arhizus during early steps of in vitro culture detected by liquid chromatography-mass spectrometry that might be responsible for the positive effect on root growth will be also presented

Mycorrhiza-like structures in rooted microshoots of Pinus pinea L.

Pinus pinea L. (stone pine) is one of the major plantation species in Iberian Peninsula, being Portugal the largest edible seed producer in the world. The induction and improvement of in vitro rhizogenesis of microshoots of Pinus pinea was developed in our laboratory using a co-culture system with ECM fungi. In the acclimation phase in mixed substrates, or in rhizotrons, anatomical and morphological studies were done to observe the evolution of the root system in microshoots from the co-culture system vs. control plants. Extensive dichotomous and coralloid branching of lateral roots occurred spontaneously in inoculated and control plants as well. Moreover, similar branching occurred in liquid culture of excised seedling roots without the presence of ECM fungi. The striking similarity of these organs with pine ectomycorrhizas prompted their anatomical analysis; however the presence of Hartig net was not confirmed. These results suggested that the development of ECM-like structures might have occurred spontaneously.info:eu-repo/semantics/publishedVersio

O-coumaric acid ester, a potential early signaling molecule in Pinus pinea and Pisolithus arhizus symbiosis established in vitro

During ectomycorrhizal (ECM) establishment, biochemical signals lead to the development of complex structures in both the plant and the fungus that ultimately result in the formation of an ectomycorrhiza. The cross-talk between partners begins before physical contact. Our objective was to investigate the chemical nature of the signals during the first stages of in vitro mycorrhization of Pinus pinea with Pisolithus arhizus. For this purpose a double-phase solid liquid medium was expressly developed for the co-culture in order to simplify the extraction and further molecules analysis. O-coumaric acid ester was identified using HPLC UV and LC DAD MS on the second day of co-culture and its presence was detected for up to 10 days. These results contribute to the characterization of biochemical signals during pre-colonization involving conifer species and an ECM fungus, and demonstrate the suitability of the double-phase medium developed for the growth of both organisms and for the analysis of released chemical mediators.info:eu-repo/semantics/publishedVersio

Pisolithus arhizus (Scop.) Rauschert improves growth of adventitious roots and acclimatization of in vitro regenerated plantlets of Pinus pinea L.

Stone pine (Pinus pinea L.) is an economically important forest tree in the Mediterranean region and has been the target of breeding and selection through micropropagation mainly for its ecological and ornamental aspects. A crucial step in micropropagation is adventitious rooting of microshoots, which often is highly inefficient in most conifer species including stone pine. Hence, we conducted in vitro co-culture of Pinus pinea microshoots with the ectomycorrhizal fungus Pisolithus arhizus (isolated from natural stands) in order to promote adventitious root growth and plant survival during acclimatization. Significant differences were found in the number of branches, in the number of roots plus branches, in total length of roots, in total length of roots plus branches, in average root length and in the length of the longest root in inoculated plants during in vitro rooting compared with non-inoculated plants. The roots of inoculated plants also grew better in vermiculite and during acclimatization in a mixed substrate compared with roots of control plants resulting in the development of vigorous root system. Overall, mycorrhizal inoculation increased the survival rate of the regenerated pine

Assessing the diversity of sea beet (Beta vulgaris L. ssp. maritima) populations

Beta vulgaris L. subsp. maritima (L.) Arcang., sea beet, is a morphologically and genetically variable species, belonging to beet primary gene -pool. This crop wild relative is a valuable genetic resource for resistance improvement in beets and could play an important role in crop yield sustainability. Eleven Madeiran sea beet populations were characterized using morphological descriptors and genetic markers. Our goal was to evaluate these populations as a potential source of valuable genetic material. Morphological characterization showed a high quantitative variation among populations. Plant height and inflorescence height parameters had the highest influence in the separation of populations. Molecular analysis was performed with polymorphic SSRs to determine genetic variability between populations. Both PCA and PCoA revealed three clusters that separated the populations according to morphological and genetic traits, respectively. This study contributes to the knowledge of sea beet diversity in Madeira’s archipelago and to the perception that the islands' specific environmental conditions influence its genetic variability, making these populations a possible gene source for sugar beet breeding programsinfo:eu-repo/semantics/publishedVersio

Functional marker development from AOX genes requires deep phenotyping and individualized diagnosis

The development of new ‘deep phenotyping’ techniques for functional markers (FM) development on alternative oxidase (AOX) gene sequences are expected to greatly increase the efficiency of association studies between the candidate FM sequences and the desired phenotype. However, it is critical to perform these studies in the appropriate target tissue/cell at the correct time point. AOX genes, due to their diversity and with differential methylation marks, are likely also subjected to such interplay between sequence and regulatory mechanisms. Polymorphisms in coding sequences may directly affect protein function, but expression regulatory switches are more abundant in non-coding regions. Transgenic technology continues to contribute to crop improvement programme, if efforts are directed more towards FM-assisted plant breeding. Hence, AOX can be put into best use if a dual approach involving genetic transformation and conventional plant breeding go hand in hand