María Elena González-Benito

Effect of exogenous ABA on embryo maturation and quantification of endogenous levels of ABA and IAA in Quercus suber somatic embryos

Knowledge of the relationship between indole-3-acetic acid (IAA) and abscisic acid (ABA) is relevant to control the development and the maturation of cork oak (Quercus suber L.) somatic embryos. The addition of 1 μM ABA to the culture medium significantly promoted somatic embryo maturation and increased both fresh and dry matter without affecting the relative water content. This effect was parallel to the pattern of variation observed in the endogenous ABA level, which increased from the immature to the mature stage. Endogenous ABA content during the occurrence of secondary embryogenesis was similar to that of the immature stage, showing that embryos with lower ABA levels produced secondary embryos. In contrast, IAA showed the highest concentration during early embryo development and decreased afterwards. Only in somatic embryos subjected to 1-week desiccation followed by stratification at 4 °C for 2 weeks, was a moderate increment of endogenous IAA content observed. IAA and ABA showed opposite levels during the development and maturation of cork oak somatic embryos and characterised specific stages of the embryonic development.

Improved somatic embryogenesis of grapevine (Vitis vinifera) with focus on induction parameters and efficient plant regeneration

A study of four parameters (induction medium, floral explant, developmental stage and year) was carried out to determine the best combination for the embryogenesis induction of eight grapevine (Vitis vinifera L.) cultivars. Anthers and ovaries were extracted from flower buds at three developmental phases and incubated in two induction media over two consecutive years. As average, the percentage of embryogenesis on Nitsch and Nitsch-derived medium (9.1%) was higher than in Murashige and Skoog-derived medium (5.9%) and embryogenesis from ovaries (10.1%) was 2-fold higher than from anthers (4.9%). Earlier flower developmental stages (II–III) favored embryogenic induction from anthers, while later stages (III–V) did it from ovaries. Induction of embryogenic cultures was genotype dependent. Two years after the establishment of the embryogenic lines, an average of 48.0% of the pro-embryogenic masses were viable and suitable to initiate cell suspensions. Embryogenic cultures of four genotypes showed a high percentage of conversion from embryos to plants: Albariño (61.8%), Garnacha (48.8%), Tempanillo (71.0%) and Sultanina (69.0%). Moreover, cell suspensions were competent for transient transformation based on β-glucuronidase assay, as up to 6,387 blue spots per Petri plate after Biolistic bombardment were obtained. Here, we present the advantage of ovaries over anthers for the embryogenesis induction of several grapevine cultivars. This is the first report of embryogenesis from the cultivars Albariño, Verdejo and Muscat Hamburg as well as transient transformation of Albariño and Tempranillo.

Effect of water content and temperature on seed longevity of seven Brassicaceae species after 5 years of storage

Maximising seed longevity is crucial for genetic resource preservation and longevity of orthodox seeds is determined by environmental conditions (water content and temperature). The effect of water content (down to 0.01 g·H₂O·g(-1) ) on seed viability was studied at different temperatures for a 5-year storage period in taxonomically related species. Seeds of seven Brassicaceae species (Brassica repanda, Eruca vesicaria, Malcolmia littorea, Moricandia arvensis, Rorippa nasturtium-aquaticum, Sinapis alba, Sisymbrium runcinatum) were stored at 48 environments comprising a combination of eight water contents, from 0.21 to 0.01 g·H₂O·g(-1) DW and six temperatures (45, 35, 20, 5, -25, -170 °C). Survival curves were modelled and P50 calculated for those conditions where germination was reduced over the 5-year assay period. Critical water content for storage of seeds of six species at 45 °C ranged from 0.02 to 0.03 g·H₂O·g(-1) . The effect of extreme desiccation at 45 °C showed variability among species: three species showed damaging effects of drying below the critical water content, while for three species it was neither detrimental nor beneficial to seed longevity. Lipid content could be related to longevity, depending on the storage conditions. A variable seed longevity response to water content among taxonomically related species was found. The relative position of some of the species as long- or short-lived at 45 °C varied depending on the humidity at which storage behaviour was evaluated. Therefore, predictions of survival under desiccated conditions based on results obtained at high humidity might be problematic for some species.

Biochemical changes induced in seeds of Brassicaceae wild species during ageing

The aim of the present study was to determine whether the loss of seed germination capacity and vigour in seeds of four wild Brassicaceae species (Brassica repanda, Moricandia arvensis, Rorippa nasturtium-aquaticum and Sinapis alba) during ageing at 45°C and 90% relative humidity was related to changes in lipid peroxidation and membrane integrity. For all of the species, ageing reduced the final germination percentage and increased the length of time required to reach 50% of final germination (T50). Large differences in longevity were observed among the species. The times required for viability to be reduced to 80 and 50% of maximum germination (P80 and P50) were the lowest for B. repanda, and these values were two times longer for M. arvensis and R. nasturtium-aquaticum and five times longer for S. alba compared with B. repanda. A loss of seed viability was not associated with malondialdehyde accumulation, suggesting that lipid peroxidation did not cause seed deterioration under these conditions. However, the conductivity test effectively detected seed deterioration in these wild Brassicaceae species, and membrane permeability correlated with both germination and vigour loss. This correlation may provide a valuable mean for early detection of seed viability in wild Brassicaceae species.

Approaches for In Vitro Conservation of Woody Plants Germplasm

The conservation of plant genetic diversity aims at preserving as much as possible extant species by using innovative and complementary approaches to guarantee the effectiveness of the safeguarding strategies and to face present problems and future threats. The development and implementation of different in vitro conservation techniques have provided improvements for the international exchange of germplasm, for the storage of different in vitro culture forms and for products generated by biotechnology. These methods are also a valuable alternative to relieve the need of large lands extensions, where reserve collections of trees belonging to many woody species are traditionally kept. This chapter provides information of several study cases, describes some useful protocols, and aims at presenting a brief overview of currently available techniques for in vitro conservation to medium- and long-term of woody plant germplasm.

Estimation of genetic diversity in seedlings of Plantago algarbiensis, an endangered species endemic to the south of Portugal in risk of global extinction

Plantago algarbiensis samp is an endangered species endemic to the Algarve, in southern Portugal. In the present work, the genetic diversity of three populations was assessed by RAPD markers. Samples were amplified using ten primers that generated 145 markers, 80% of which were polymorphic. Tunes population presented the highest polymorphism percentage (73.68%) and Algoz the lowest (67.67%). In the cluster analysis, two major groups were formed, one including individuals from Gambelas and the other clustered together individuals from the other populations. The highest level of genetic diversity, estimated by both Nei’s gene diversity and Shannon’s information measure, was found in Tunes and the lowest in Algoz. From the estimates of Shannon’s index, the proportion of the diversity within populations was 86.12% and that among populations was 13.88%. Similar results were obtained by AMOVA analysis. A correlation was found between geographic distance and genetic differentiation among populations. The Nm values obtained suggest a high level of gene flow among populations, which was inversely proportional to the distance between populations. RAPDs proved to be a useful tool to determine the genetic diversity of P. algarbiensis populations. The data obtained can be used to develop effective conservation strategies to prevent the decline of populations.