Armando Costa

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

SummarySomatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl−1 sucrose, 3 gl−1 gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.

Acclimatization of secondary somatic embryos derived plants of Eucalyptus globulus Labill.: an ultrastructural approach

This paper reports the complete process from secondary emblings (SE-derived plants) regeneration to acclimatization of Eucalyptus globulus and describes histocytological changes that occur in leaves from in vitro to ex vitro acclimatization for a 3-month period. After elongation, plants were transferred to pots with sterilized peat:perlite and acclimatized in a phytotron, with progressive reduction of RH and increase of light intensity. Histocytological analyses were performed in fixed material using light microscopy and ultrastructural changes followed by electron microscopy (SEM and TEM). The protocol used allowed the successful acclimatization of the emblings. Plants looked morphologically normal and FCM screening revealed no ploidy or DNA content abnormalities. Histocytological analyses showed significant changes along time, mostly in stomata shape and aperture, starch reserves, chloroplast morphology and mesophyll differentiation. This is the first report concerning emblings acclimatization to ex vitro conditions in Eucalyptus. It was clearly demonstrated that during acclimatization emblings suffered profound changes in leaf morphology in order to successfully adapt to ex vitro conditions.

Large-scale field acclimatization of Olea maderensis micropropagated plants: morphological and physiological survey

Micropropagation allows large-scale plant multiplication and germplasm preservation, representing an added value in forest breeding strategies to combat desertification and/or protect endangered species. We developed a large-scale micropropagation protocol of Olea maderensis (a native endangered wild olive of Madeira Archipelago) using OMG medium (rich in Fe, Mg and Mn) supplemented with zeatin for elongation and with NAA for rooting. We now describe the performance of micropropagated plants during five-period field acclimatization: (1) in vitro, (2) growth-cabinet, (3) greenhouse, (4) open-greenhouse, and (5) field mountain in Porto Santo Island. One hundred OG4 plants were acclimatized, showing >95% surviving rates. During acclimatization, several physiological parameters were evaluated; water content remained higher in in vitro/greenhouse conditions, decreased in field leaves. Soluble protein contents decreased during the first acclimatization periods increasing thereafter. Membrane permeability slightly increased during the field acclimatization. Chlorophylls content increased in in vitro leaves, while during acclimatization, mostly chl b decreased, increasing chl a/chl b ratio. F0 decreased in first acclimatization periods, increasing thereafter, while the other parameters (Fv; Fm; Fv/Fm) decreased. Nutrient contents decreased in plants transferred to poor field soil conditions, reaching values similar to mother plant leaves. Overall, with the exception of PSII fluorescence, field acclimatized plants had similar values to mother plants, showing a good adjustment to stressful field conditions. This protocol is being used in large-scale micropropagation within a reforestation program, and is an example of R&D technologies with immediate application on protection of endangered ecosystems.

Chloroplast functionality assessment by flow cytometry: Case study with pea plants under Paraquat stress

Photosynthesis is one of the most important processes in plant biology and in the development of new methodologies that allow a better understanding and characterization of the photosynthetic status of organisms, which is invaluable. Flow cytometry (FCM) is an excellent tool for measuring fluorescence and physical proprieties of particles but it has seldom been used in photosynthetic studies and thus the full extent of its potentialities, in this field of research, remains unknown. To determine the suitability of FCM in photosynthesis studies, pea plants were exposed to Paraquat and their status was analyzed during 24 h. FCM was used to evaluate the integrity (volume and internal complexity) and the relative fluorescence intensity (FL) of chloroplasts extracted from those plants. To elucidate which type of information the FL conveys, FL values were correlated with the minimum fluorescence level (F0), maximum fluorescence level (Fm) and maximum photochemical efficiency of PSII (Fv/Fm), obtained by using Pulse-Amplitude-Modulation (PAM) fluorometry. Results indicate that: (1) the biomarkers used to evaluate the structural integrity of the chloroplasts were more sensitive to Paraquat exposure than the ones related to fluorescence; (2) the variation of the chloroplast’s structure, as time progressed, pointed to a swelling and subsequent burst of the chloroplast which, in turn, compromised fluorescence emission; (3) FL presented a high and significant correlation with the Fv/Fm and to a lesser degree with Fm but not with F0; (4) pigment content did not reveal significant changes in response to Paraquat exposure and is in agreement with the proposed model, suggesting that the cause for fluorescence decrease is due to chloroplast disruption. In sum, FCM proved to be an outstanding technique to evaluate chloroplastidal functional and structural status and therefore it should be regarded as a valuable asset in the field of photosynthetic research.