Augusto Peixe

Induction of Haploid Morphogenic Calluses from in vitro Cultured Anthers of Prunus Armeniaca cv. ‘Harcot’

Apricot (Prunus armeniaca) ‘Harcot’ anthers, were cultured in vitro for the production of haploid plants. The best androgenic response was achieved with Nitsch and Nitsch (1969) medium, supplemented with 4.52 μM 2,4-D, 4.52 μM zeatin, 2.85 μM IAA and 40 g l−1 sucrose. Cultures were maintained in the dark for 8 days, at 28°C, followed by transfer to a 16-h photoperiod, with 35 μm m−2 s−1 light intensity and 24/22°C day/night temperature. The androgenic response was correlated with the floral bud size, its phenologic stage and the level of microspore evolution. Anthers containing microspores at the tetrad/uninucleate stage were the most appropriate. The ploidy level of the calluses was evaluated by flow cytometry revealing that they range from haploid to octaploid. Mixoploid calluses have also been identified. Histological studies showed that the haploid calluses have their origin in the microspores. Nodular structures consisting of cells with dense cytoplasm and differentiated xylem elements were observed and were surrounded by an autofluorescent layer, probably due to cutin deposition.

Gamma-irradiated pollen induces the formation of 2n endosperm and abnormal embryo development in European plum (Prunus domestica L., cv. “Rainha Cláudia Verde”)

The effect of gamma radiation on pollen germination capacity and pollen tube growth was evaluated in vitro and in situ conditions. In vitro experiments, revealed that irradiation significantly affects pollen viability, mainly for levels higher than 200 Grays (Gy). Also, for levels higher than 200 Gy, in situ observations showed that no pollen tube reached the ovule. Fruit set results confirmed that for irradiation levels higher than 200 Gy, all fruits dropped before 90 days after pollination (DAP). Most of the seeds obtained from 200 Gy pollination treatments were empty. Other seeds contained only endosperm or endosperm and embryos with abnormal development. For those seeds, flow cytometry analysis revealed sometimes the presence of a 2n endosperm, indicating that double fertilization did not occur and leading to the possibility of haploid embryo formation.

Involvement of alternative oxidase (AOX) in adventitious rooting of Olea europaea L. microshoots is linked to adaptive phenylpropanoid and lignin metabolism

Alternative oxidase (AOX) has been proposed as a functional marker candidate in a number of events involving cell differentiation, including rooting efficiency in semi-hardwood shoot cuttings of olive (Olea europaea L.). To ascertain the general importance of AOX in olive rooting, the auxin-induced rooting process was studied in an in vitro system for microshoot propagation. Inhibition of AOX by salicylhydroxamic acid (SHAM) significantly reduced rooting efficiency. However, the inhibitor failed to exhibit any effect on the preceding calli stage. This makes the system appropriate for distinguishing dedifferentiation and de novo differentiation during root induction. Metabolite analyses of microshoots showed that total phenolics, total flavonoids and lignin contents were significantly reduced upon SHAM treatment. It was concluded that the influence of alternative respiration on root formation was associated to adaptive phenylpropanoid and lignin metabolism. Transcript profiles of two olive AOX genes (OeAOX1a and OeAOX2) were examined during the process of auxin-induced root induction. Both genes displayed stable transcript accumulation in semi-quantitative RT-PCR analysis during all experimental stages. In contrary, when the reverse primer for OeAOX2 was designed from the 3′-UTR instead of the ORF, differential transcript accumulation was observed suggesting posttranscriptional regulation of OeAOX2 during metabolic acclimation. This result confirms former observations in olive semi-hardwood shoot cuttings on differential OeAOX2 expression during root induction. It further points to the importance of future studies on the functional role of sequence and length polymorphisms in the 3′-UTR of this gene.Key message The manuscript reports the general importance of AOX in olive adventitious rooting and the association of alternative respiration to adaptive phenylpropanoid and lignin metabolism.

Use of morphometric parameters for tracking ovule and microspore evolution in grapevine (Vitis vinifera L., cv. “Aragonez”) and evaluation of their potential to improve in vitro somatic embryogenesis efficiency from gametophyte tissues

Somatic embryogenesis induction from in vitro cultured stamens and carpels is highly dependent on explants’ inoculation at specific developmental stages. To establish good correlations between measurable morphometric parameters of flowers or flower buds and developmental stages of micro- and macrosporogenesis, this procedure is the easiest way to simplify the in vitro culture procedures. These correlations were established here for the most important Iberian grapevine cultivar, the “Aragonez”, named “Tempranillo” in Spain and “Tinta Roriz” in the north of Portugal, and were based in floral buds and anther measurements. The anther length, with a correlation coefficient of 0.90, proved to be the best morphometric parameter to follow microsporogenesis evolution. A correlation between micro- and macrosporogenesis evolutionary stages was also positively established, allowing the use of morphometric parameters for tracking ovule evolution as well. Carpels in several evolutionary stages were in vitro cultured to evaluate the aging effect on the capacity for somatic embryogenesis induction. Explants inoculated in the earliest stages of macrosporogenesis presented the best results. Media culture formulations were also tested for ovary culture, with the best results being achieved with a 5:1 auxin/cytokinin ratio.

AOX1-Subfamily Gene Members in Olea europaea cv. “Galega Vulgar”—Gene Characterization and Expression of Transcripts during IBA-Induced in Vitro Adventitious Rooting

Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar ”Galega vulgar”. The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars.