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- Cryopreservation as a tool for preserving genetic variability of endangered species endemic from Algarve regionPublication . Coelho, Natacha; Romano, Anabela; González-Benito, María ElenaThis thesis aims the development of complementary conservation strategies based on plant biotechnology to support the preservation of three rare endemic species of the Algarve, Portugal. The species studied were Thymus lotocephalus G. López & R. Morales, Plantago algarbiensis Samp. and Tuberaria major (Willk.) P. Silva & Rozeira. An in vitro propagation protocol was firstly developed for T. lotocephalus using seedlings as explants (Chapter 2.1). The germination conditions and feasibility of cryopreservation was also studied for T. lotocephalus seeds (Chapter 2.2). In Chapter 2.3 a shoot tip cryopreservation protocol was developed for this species and the genetic stability of the cryopreserved material was assessed. For P. algarbiensis, the work was initiated with the cryopreservation of seeds and nodal segments (Chapter 3.1), followed by the study of the genetic diversity of three wild populations (Chapter 3.2). Chapter 4.1 was dedicated to the improvement of the germination and cryopreservation of T. major seeds and the development of a shoot tip cryopreservation protocol. An efficient micropropagation protocol was established for the mass production of T. lotocephalus (Chapter 2.1) and the seeds germination requirements were determined (Chapter 2.2). From the shoots produced in vitro, apices were excised and used in the development of a shoot tip cryopreservation protocol (Chapter 2.2). P. algarbiensis nodal segments were successfully cryopreserved using two different methods (Chapter 3.1). The structure of P. algarbiensis populations was effectively evaluated using molecular markers, revealing high levels of genetic diversity within populations (Chapter 3.2). Cryopreservation proved to be a suitable method for the conservation of seeds from both species (Chapters 2.2 and 3.1). Finally, the germination conditions and cryopreservation of T. major seeds were considerably improved and the cryopreservation of T. major shoot tips was accomplished without major optimizations of the different procedures (Chapter 4.1). In conclusion, during the course of this thesis different ex situ conservation strategies were developed giving a major contribution to the preservation of T. lotocephalus, P. algarbiensis and T. major.
- In vitro propagation of insectivorous plants for phytochemical purposesPublication . Coelho, Natacha; Romano, Anabela; Gonçalves, SandraThe aims of this research work were to establish in vitro propagation protocols for the insectivorous plants D. intermedia, D. rotundifolia and P. vulgaris, using seeds as explants, in order to produce plants for biocompound extraction and to replenish natural populations. The antioxidant capacity of the extracts prepared from in vitro cultures of these species was evaluated. The establishment of a simple protocol for in vitro propagation of D. intermedia is reported. Protocols for micropropagation of P. vulgaris and D. rotundifolia were initiated but still need to be improved. Cultures of all three species were initiated from shoots of in vitro produced seedlings. The effect of three MS medium concentrations, ¼MS, ½MS and MS, without PGR were tested in D. intermedia and P. vulgaris. The same media supplemented with 0.1 mg l-1 KIN were investigated in D. intermedia and ¼MS supplemented with 0.5 mg l-1 KIN or ZEA were investigated in P. vulgaris. In all media D. intermedia shoots rooted during the proliferation phase, presenting a multiplication and rooting percentage above 90%. The highest mean number of shoots was attained in ¼MS and ¼MS supplemented with 0.1 mg l-1 KIN. All the D. intermedia plants were successfully acclimatized to ex vitro conditions. The formation of roots also occurred during P. vulgaris proliferation phase, though with poorer results for both proliferation and rooting. Nevertheless, the best results were obtained in the media supplemented with cytokinins. In D. rotundifolia the media tested were ¼MS and MS. Multiplication percentage and mean number of shoots was higher in ¼MS medium and no rooting was observed. Antioxidant activity of D. intermedia, D. rotundifolia and P. lusitanica extracts was measured using three different methods: ORAC, F-C phenolics and TEAC. Methanol and standard maceration improved the efficiency of the antioxidant extraction procedure. For all tested methods P. lusitanica presented the highest antioxidant activity, representing a possible good source of antioxidants.