Browsing by Author "González-Benito, María Elena"
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- Approaches for the cryopreservation of Plantago algarbiensis, a rare endemic species of the AlgarvePublication . Coelho, Natacha; González-Benito, María Elena; Romano, AnabelaPlantago algarbiensis is an endangered endemic species from the Algarve, Portugal. The main goal of this study was to investigate the viability of cryopreservation procedures in the conservation of seeds and nodal explants from this species. Seeds were directly immersed in liquid nitrogen (LN) for 30 days. Two methods were tested for the cryopreservation of nodal explants, namely droplet-vitrification and encapsulation-dehydration. For both methods, nodal segments were precultured on Murashige and Skoog (MS) medium and recovered on MS supplemented with 0.2 mg l(-1) 6-benzyladenine (BA), after freezing. After 30 days in LN, the germination capacity of seeds was not affected. The regrowth percentages of cryopreserved nodal segments were approximately 60%. With the droplet-vitrification method, a regrowth percentage of 60.0+/-15.2% was obtained after 120 min exposure to PVS2 (plant vitrification solution 2) and with encapsulation-dehydration method the highest percentage, 63.3+/-9.6%, was achieved after 3 h desiccation. Seed cryopreservation and cryopreservation of nodal segments by droplet-vitrification and encapsulation-dehydration are therefore effective approaches for the conservation of P. algarbiensis.
- Cryopreservation of shoot tips from the endangered endemic species Tuberaria majorPublication . Coelho, Natacha; González-Benito, María Elena; Romano, AnabelaTuberaria major is an endangered endemic species from the Algarve, in the south of Portugal. We investigated two techniques for the cryopreservation of T. major shoot tips, namely vitrification and encapsulation-dehydration. Before the cryopreservation trials, shoot tips were precultured for 1 day on liquid Murashige and Skoog (MS) medium containing 0.3 M sucrose. For the vitrification method, shoots tips were exposed for 0, 30, 60, 90 and 120 min to plant vitrification solution 2 (PVS2). As for the encapsulation-dehydration method, shoot tips were dried inside a laminar air flow cabinet for 0, 1, 2, 3, 4, 5 and 6 h at room temperature. The highest regrowth percentages were approximately 60 and 67 % for vitrification and encapsulation-dehydration, respectively. The best times were 60 min exposure to PVS2 for vitrification and 3 h desiccation for encapsulation-dehydration. Though these are preliminary results, the use of the cryopreservation techniques tested here proved to be an important asset in the conservation of this endangered species and will complement the conservation strategies previously developed.
- Establishment of an in vitro propagation protocol for Thymus lotocephalus, a rare aromatic species of the Algarve (Portugal)Publication . Coelho, Natacha; Gonçalves, Sandra; González-Benito, María Elena; Romano, AnabelaThe aim of this work was to develop an in vitro propagation protocol for the endangered species Thymus lotocephalus using seedlings as explants. Several macronutrient concentrations of Murashige and Skoog medium (MS), cytokinin types and concentrations, and cytokinin/auxin combinations were tested to assess the shoots’ proliferation capacity. Although the best proliferation results were obtained with 6-benzyladenine, high percentages of hyperhidric shoots were observed. Because high proliferation of healthy shoots was observed in MS medium that was free of plant growth regulators, this medium was chosen for proliferation studies. The best rooting results were achieved in MS medium without auxins (92.00 ± 6.11%, 6.54 ± 0.52 and 1.60 ± 0.10 cm regarding rooting frequency, number of roots per shoot and longest roots, respectively) or supplemented with 0.5 mg l-1 indole-3-acetic acid (98.00 ± 2.11%, 11.14 ± 0.75 and 2.40 ± 0.24 cm, respectively). Plantlets were successfully acclimatised to ex vitro conditions with a survival rate of 93.33%. With the development of this micropropagation protocol, an important contribution has been made to the conservation of the endangered species T. lotocephalus.
- Germination and cryopreservation tolerance of seeds from the rare aromatic species Thymus lotocephalusPublication . Coelho, Natacha; Gonçalves, Sandra; González-Benito, María Elena; Romano, AnabelaThymus lotocephalus G. López and R. Morales is a rare aromatic species endemic from the south region of Portugal. We investigated the germination behavior and the cryopreservation tolerance of T. lotocephalus seeds collected from four populations. Seeds were set to germinate at 15 or 25 ◦C under light or darkness. The best germination temperature tested was 15 ◦C, under either light or darkness, with final germination percentages above 80% and mean germination time (MGT) below 10 days. The results showed that T. lotocephalus has non-dormant seeds. Seeds from different populations presented few differences on the final germination percentage and MGT. To assess seed tolerance to cryopreservation, they were directly immersed in liquid nitrogen for 30 days. Overall, we found that cryopreservation was non-detrimental to germination of T. lotocephalus seeds, thus being a reliable method for germplasm conservation of this endangered species.
- Germination requirements and cryopreservation tolerance of seeds of the endangered species Tuberaria majorPublication . Gonçalves, Sandra; Fernandes, Laura; Pérez-García, Felix; González-Benito, María Elena; Romano, AnabelaThe germination requirements and the cryopreservation tolerance of Tuberaria major (Cistaceae) seeds, an endangered plant species endemic from the Algarve region (South of Portugal) were studied. The final germination percentage of untreated seeds was very low (less than 2%) and was significantly improved by heat treatments (maximum of 54%). No significant differences were observed among the germination percentages reached by seeds submitted to boiling water or dry heat (100°C for different periods: 15, 30 and 60 min). Moreover, the temperature regime did not affect the final germination percentage. Cryopreservation did not affect the germination rate in comparison with the control, although the heat treatment was still necessary after cryopreservation. In conclusion, it was observed that heat stimulated germination of T. major seeds and cryopreservation could provide a method for long-term storage of germplasm of this endangered species.