Relatório de Consultadoria Técnico-Científico INNEA-Alternativas Biotecnológicas Valladolid, Espanha

Entre os dias 30 de janeiro e 4 de fevereiro de 2018 foi realizada uma consultadoria técnico-científica a empresa espanhola INNEA Alternativas Biotecnológicas, com sede social na cidade de Valladolid, Espanha. Esta empresa consta de um laboratório de cultura in vitro de espécies lenhosas na localidade de Dueñas, Palencia, onde efetivamente decorreram os trabalhos. O objetivo da visita foi prestar um serviço de consultadoria na cultura in vitro de espécies lenhosas de interesse comercial para esta empresa espanhola, particularmente na micropropagação do pinheiro manso (Pinus pinea L.). O convite foi efetuado pela experiência que a equipe da Universidade de Évora formada por formada por docentes do Departamento de Biologia (Professores Amely Zavattieri, Celeste Santos Silva, Luís Silva Dias, Paulo de Oliveira e Isabel Pereira† ) e do Departamento de Química (Professores Ana Teresa Caldeira, Rosário Martins, Dora Teixeira), dos aluno de doutoramento Rogério Louro, dos doutores Carla Ragonezi e Mário Rui Castro, técnicos superiores do Departamento de Biologia (Otília Miralto e Elsa ganhão) e consultora externa Krystyna Klimaszewska do Forest Service, Canada, possuem na organogénese de pinheiro manso a partir de cotilédones de sementes maturas e a micorrização in vitro com fungos ectomicorrízicos. Estes trabalhos que decorreram por mais de uma década, contaram com a dedicação de uma numerosa equipa de investigação. Entre outros motivos, o objeto da visita foi também falar da patente a as suas possibilidades de aplicação comercial. O nosso sistema de co-cultura foi patenteado (PT 105239, INPI) pelo grande interesse para aplicação entre as co-culturas plantas-fungos e para a realização de diversos estudos bioquímicos das relações plantas simbióticas, esta patente recebeu reconhecimento internacional, em este caso particular foi um convite para realizar uma consultadoria numa empresa do setor da produção de plantas

Relatório de Actividades - Sabática 2015

Relatório das actividades realizadas na Estação do Instituto Nacional de Tecnologia Agropecuária (INTA), Bella Vista, Corrientes, Argentina no ano de 201

Histological Studies of Mycorrhized Roots and Mycorrhizal-Like-Structures in Pine Roots

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

Adventitious rooting of conifers: influence of biological factors

Vegetative propagation of superior conifer trees can be achieved e.g. through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tissue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots including physical and chemical ones such as plant growth regulators, carbohydrates, light quality, temperature and rooting substrates or media (reviewed by Ragonezi et al. 2010). The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant- growth-promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microorganisms could contribute not only to adventitious root development but also help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests

Effects of carbon source, carbon concentration and culture conditions on in vitro rooting of Pinus pinea L. microshoots

In stone pine (Pinus pinea L.), clonal propagation via adventitious shoot formation from cotyledons has been reported before but rooting of these shoots is poor. The number of rooted shoots had low frequency limiting the used of micropropagation protocols as a choice for mass propagation of superior genotypes. Therefore the main objective of the present work was to increase the number and quality of roots per shoot in order to ensure the survival and growth of a great number of plants in the acclimation phase. To achieve this, different combinations of carbon source (sucrose or glucose) at different concentrations, under different environmental conditions (temperature and light), were tested in the induction and expression phases of the adventitious root formation. Shoots of different clones obtained via organogenesis have been employed for the experiments. Observations were made on the rooting percentage, root length and number of roots per shoot. The results showed a general increased of the number of roots per shoot and an earlier root formation when glucose was used as a carbon source. However, there were no differences in the percentage of rooting between the carbons sources tested. The best results were obtained using 0.117 M of glucose and dark treatment combined with 19°C during the induction phase of the rhizogenic process. Light and low sugar concentration proved to be beneficial for the expression phase, increasing the root length. A remarkable interclonal difference in the ability to form roots was observed. It was possible to obtain a rooting percentage of more than 75% in several of the tested clones

Influence of light quality and intensity on adventitious root formation in microshoots of Pinus pinea L.

In the present study of Pinus pinea L., further improvement of microshoot rooting was achieved by applying Cool-white light at increased intensity from 60 to 90 µmol m-2 s-1. In contrast, light provided by Gro-lux lamps promoted rooting of the microshoots at the same frequency regardless of its intensity. Majority of microshoots (70.4%) grown under Cool-white lamps at the intensity of 90 µmol m-2 s-1 were also significantly taller when compared with those from other tested treatments

Induction of somatic embryogenesis as an example of stress-related plant reactions

In this review, we address the role of stress as one of the principal causes for a cell or tissue to change its preexisting somatic program, reprogramming itself to express the embryogenic pathway. The focus of this paper is the effect of different stress conditions on the induction phase of plant somatic embryogenesis, as well as the development of embryogenic competence as a result of the applied stresses. We also present a variety of data that link plant somatic embryogenesis, DNA methylation and oxidative stress response

The gymnosperm Pinus pinea contains both AOX gene subfamilies, AOX1and AOX2

The gymnosperm Pinus pinea L. (stone pine) is a typical Mediterranean pine used for nuts and timber production,and as an ornamental around the world. Pine genomes are large in comparison to other species. The hypothesis that retrotransposons, such as gymny, made a large contribution to this alteration in genome size was recently confirmed. However, P. pinea is unique in other various aspects. P. pinea demonstrates a different pattern of gymny organization than other Pinus subgenera. Additionally, P. pinea has a highly recalcitrant behaviour in relation to standard conifer protocols for the induction of somatic embryogenesis or rooting. Because such types of cell reprogramming can be explained as a reaction of plant cells to external stress, it is of special interest to study sequence peculiarities in stress-inducible genes, such as the alternative oxidase (AOX). This is the first report containing molecular evidence for the existence of AOX in gymnosperms at the genetic level. P. pinea AOXs were isolated by a polymerase chain reaction (PCR) approach and three genes were identified. Two of the genes belong to the AOX1 subfamily and one belongs to the AOX2 subfamily. The existence of both AOX subfamilies in gymnosperms is reported here for the first time. This discovery supports the hypothesis that AOX1 and AOX2 subfamilies arose prior to the separation of gymnosperms and angiosperms, and indicates that the AOX2 is absent in monocots because of subsequent gene loss events. Polymorphic P. pinea AOX1 sequences from a selected genetic clone are presented indicating non-allelic, non-synonymous and synonymous translation products

Adventitious rooting of conifers: influence of physical and chemical factors

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

Alqueva hydro-meteorological experiment(alex): first results of aquatic ecological assessment

The ALqueva hydro-meteorological EXperiment (ALEX) field campaign took place monthly during summer 2014 and consisted in in situ measurements and sampling of water and biological elements, collected from three fixed platforms placed in the lacustrine zone. This integrated overview, including meteorological, environmental and biological results contributes to improve the knowledge of the reservoir dynamics and therefore to propose adequate management measures to preserve the observed biological integrity