Several studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots.

Na natureza, as plantas e os microrganismos estabelecem associações de várias ordens. Nas culturas in vitro de plantas, mesmo as associações favoráveis com microrganismos foram, durante muitos anos, consideradas como contaminantes. Só mais tarde, as vantagens da inoculação in vitro (co-cultura) foram demonstradas e as técnicas de biotização (bacterização ou micorrização) usadas com o objetivo de melhorar as condições de crescimento in vitro. As dificuldades do enraizamento in vitro de uma das espécies mais importantes da floresta mediterrânica portuguesa, Pinus pinea L., conduziu à escolha deste sistema biológico, como matéria de estudo para a tese. Neste estudo, foram utilizados fungos ectomicorrízicos para otimizar a fase de enraizamento de plantas de Pinus pinea L. micropropagadas via organogénese. A introdução de ectomicorrízas no processo de micropropagação reativou o crescimento das raízes e induziu a melhoria dos vários parâmetros do sistema radicular adventício conduzindo a uma menor perda de plantas durante a aclimatização. Com efeito, a micorrização melhorou a funcionalidade das raízes, facilitando a absorção de nutrientes e de água. Neste trabalho, efetuou-se também uma extensiva caracterização morfológica e molecular das ectomicorrízas associadas a P. pinea. Das várias co-culturas testadas, selecionou-se a interação Pisolithus arhizus/P. pinea para estudar os sinais bioquímicos pré-simbióticos estabelecidos durante as etapas iniciais da co-cultura in vitro. Para possibilitar este estudo foi desenvolvido um novo sistema de co-cultura, o qual já está patenteado. Os resultados indicam que a presença de compostos fenólicos, nomeadamente o ácido o-coumarico, poderão ser importantes mediadores na interação fungo/planta.

Beta patula Aiton is a crop wild relative (CWR) which belongs to the Gene Pool 1b and is considered a Critically Endangered species, and is present in very specific environments, such as the Desembarcadouro islet (DI) in Ponta de São Lourenço or Chão islet (CI) in the Desertas Islands. The ISOPlexis Center (University of Madeira) has been providing continuous support for its in situ conservation by keeping a management plan of wild populations and an ex situ conservation strategy through the storage of accessions in the ISOPlexis GeneBank. The present work intends to present the spatial distribution of genetic variability and diversity in these B. patula populations using eight polymorphic Simple Sequence Repeat (SSR) markers. The overall results lead to the identification of three spots with a high genetic diversity. CI with a small cluster of individuals shows a genetic footprint different from DI, having unique alleles present in its population. DI has two distinct areas: the western area, with a higher individual density but with a lower genetic diversity and higher allele fixation; and the central area, with a lower individual count but with a higher genetic diversity and with the presence of unique alleles. Despite some genetic differences, the comparison of the two islets’ DI and CI populations shows that they have more similarities than differences. Analysis of the Molecular Variance, based on the hierarchical cluster, showed a 9% diversity between populations, 68% among individuals, and 23% within individuals. This data will be used for the establishment of a protocol to monitor and manage B. patula genetic diversity under a genetic reserve, subsequently contributing to the European Genetic Reserve network implementation and the protection of this important CWR.

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have benefi cial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were col lected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporo carps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effective ness of the techniques used to amplify DNA polymorphic sequences, which enhances the characte rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.

Sewage sludge (SS) is derived from wastewater treatment plants and can be used as a biofertilizer when properly stabilized. This work aimed to evaluate SS application for agricultural production improvement. SS was tested on Porto Santo Island (Portugal). The experiment was randomly designed with three 25 m2 plots for each treatment (2 SS concentrations + control without SS) and performed in two consecutive cycles. For the first cycle, dehydrated sludge was mixed with soil, obtaining final concentrations of 0.8 kg/m2 (C1) and 1.6 kg/m2 (C2). Half of the concentration was used for the second cycle. Fifty-eight sweet potato plants were used in each plot. SS application boosted the agronomic parameters of biomass, productivity, and shoot biomass. Furthermore, improvements in soil properties were observed, mainly for pH, CEC, and NO3 -N, with no significant increase in heavy metals. For the edible parts, heavy metal concentrations decreased, and Pb was the only one that still exceeded the maximum limits. The results demonstrated that SS application to low-fertility soil is effective in improving the agronomic parameters of sweet potato and enhancing soil features. Further studies considering other variables, i.e., SS origin, soil properties, and the crop, must be carried out to propose custom applications.

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have beneficial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were collected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporocarps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effectiveness of the techniques used to amplify DNA polymorphic sequences, which enhances the ­characte­rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.

Oral presentation Micropropagation of recalcitrant pine (Pinus pinea L.). An overview of the effects of ectomycorrhizal inoculation Carla Ragonezi1; Ana Teresa Caldeira2 3; Maria do Rosário Martins2,5; Otília Miralto5,6; Luís Silva Dias6; Elsa Ganhão6, Krystyna Klimaszewska4, Amely Zavattieri5,6 1 Plant Breeding and Biotechnology Laboratory, University of Évora, Ap. 94, 7002-554 Évora, Portugal 2 Department of Chemistry, University of Évora, Ap. 94, 7002-554 Évora, Portugal 3 CQE, University of Évora, Rua Romão Ramalho, nº 59, 7000-671 Évora, Portugal 4 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 10380, Stn. Sainte-Foy, Québec, QC G1V 4C7, Canada 5 ICAAM, University of Évora, Ap. 94, 7002-554 Évora, Portugal 6 Department of Biology, University of Évora, Ap. 94, 7002-554 Évora, Portugal Corresponding author: Carla Ragonezi email: cazi04@yahoo.com.br Abstract 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 utilization 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 also it is a pioneer species particularly for cork and holm oaks degraded ecosystems. The stone pine plantations are today a major source of income for forestry holdings. The 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 select 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 (both quality and duration) and temperature were used at different concentrations and levels as well as compounds such as coumarine; salicylic acid, polyamines, etc were tested for induction and expression phases of adventitious rooting. Despite these efforts, the 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 to 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 detected between Pinus pinea/Pisolithus arhizus during early steps of in vitro culture by liquid chromatography-mass spectrometry that might be responsible for the positive effect on root growth will be also presented. Key words: acclimation, co-culture, ectomycorrhiza, in vitro adventitious rooting, micropropagation, stone pine.

In conifers, vegetative propagation of superior genotypes is the most direct means for making large genetic gains, because it allows a large proportion of genetic diversity to be captured in a single cycle of selection. There are two aims of vegetative propagation, namely large-scale multiplication of select genotypes and production of large numbers of plants from scarce and costly seed that originates from controlled seed orchard pollinations. This can be achieved, in some species, either through rooted cuttings or rooted microshoots, the latter regenerated through tissue culture in vitro. Thus far, both strategies have been used but often achieved limited success mainly because of difficult and inefficient rooting process. In this overview of technology, we focus on the progress in defining the physical and chemical factors that help the conifer cuttings and microshoots to develop adventitious roots. These factors include plant growth regulators, carbohydrates, light quality, temperature and rooting substrates/media as major variables for development of reliable adventitious rooting protocols for different conifer species.

Several studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots.

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have beneficial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were collected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporocarps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effectiveness of the techniques used to amplify DNA polymorphic sequences, which enhances the ­characte­rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.