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- High-frequency in vitro propagation of the endangered species Tuberaria majorPublication . Gonçalves, Sandra; Fernandes, Laura; Romano, AnabelaA novel protocol suitable for the micropropagation of the endangered species Tuberaria major using seedlings as explants is reported. Using this protocol, we studied the effects of explant type (apical shoots and nodal segments) and cytokinins [6-benzyladenine (BA), kinetin,and zeatin (ZEA)] on shoot proliferation. Explant type significantly influenced the proliferation frequency and mean number of shoots, with nodal segments showing a higher proliferation capacity. The mean number of shoots was significantly higher when the explants were cultured in half-strength (1/2) MS medium supplemented with 0.2 mg l-1 BA (6.83 ± 0.77 shoots) or ZEA (6.55 ± 0.71 shoots). The shoots showed a great rooting capacity that was significantly influenced by the concentration of MS macronutrients but not by the concentration of auxins. The highest rooting frequencies (97–100%) were obtained in 1/2 MS medium with or without plant growth regulators. The plants obtained were easily acclimatized to ex vitro conditions, with 97% surviving after 6 weeks. The micropropagated plants were successfully reintroduced into their natural habitat and exhibited normal development. In conclusion, our culture protocol, with efficient seed germination, subsequent multiplication of nodal explants using ZEA at 0.2 mg l-1, and successful ex vitro establishment of well-rooted plantlets on 1/2 MS medium, provides a simple and reliable methodology for the large scale propagation of T. major, thereby contributing to germplasm preservation of this endangered species.
- Micropropagation of Drosophyllum lusitanicum (Dewy pine), an endangered West Mediterranean endemic insectivorous plantPublication . Gonçalves, Sandra; Romano, AnabelaIn this work, in vitro clonal propagation of Drosophyllum lusitanicum (Dewy pine) was obtained from seedlings germinated in vitro. Seeds were collected in various populations identified in the Algarve region and germinated in vitro on MS medium supplemented with 0.5 mg l 1 BA (6-benzyladenine) and 0.1 mg l 1 GA3 (gibberellic acid). The obtained shoots were used in several multiplication assays. The best results were observed in MS medium supplemented with 0.2 or 0.5 mg l 1 zeatin. The highest rooting frequency (83%) was observed on 1/4MS medium supplemented with 0.2 mg l 1 IBA (indole-3-butyric acid). Fifty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. Plans are underway to reintroduce the in vitro produced plants from this study in selected locations in their natural habitat.
- Strategies to improve rooting and acclimatization of cork oakPublication . Romano, Anabela; Martins Loução, M. A.In this work the inclusion of 2 mg l(-1) of indole-3-butyric acid (IBA) in the rooting medium produced 78% rooting. The rooting percentage was improved to 95% by basal immersion of shoots in 0.5 g l(-1) IBA for 2 min. With this method high number of long and vigorous roots developed during the second week after IBA treatment. Differences in root morphology related with rooting induction method were observed. The roots formed by dipping were more branched than those resulting by auxin addition to the culture medium. Darkness during the first week of rooting resulted in a remarkable enhancement of the rooting percentage, number of roots developed per shoot and the length of the longest root. Well-rooted plantlets were transplanted into a mixture of peat and vermiculite and incubated in a growth chamber with high humidity. Hardening of plantlets under high humidity during four weeks was found to be essential for successful acclimatization. After two months, plants were transferred to the glasshouse and the percentage of surviving plantlets after six months ranged from 60 % to 72 %.
- A new medium formulation for in vitro rooting of carob tree based on leaf macronutrients concentrationsPublication . Gonçalves, Sandra; Correia, P. J.; Martins-Loução, M. A.; Romano, AnabelaExperiments were performed to optimize the macronutrients concentrations for in vitro rooting of Ceratonia siliqua micropropagated shoots. Several dilutions of Murashige and Skoog (MS) medium were tested: full-strength MS, halfstrength MS (½MS), and ½MS + full N. The frequency of in vitro rooting was enhanced when the ½MS was used (50 % rooted shoots). Mature leaves from 20 - 30 year-old carob trees and from 2 year-old micropropagated plants were collected and the concentrations of macronutrients (N, P, K, Ca, Mg) assessed. Based on the mineral composition of the leaves a new medium was formulated and compared with the previous ones showing an increment of the rooting frequency to 80 %. Moreover, shoots rooted in the new medium did not show symptoms of apical necrosis that occurred in the other tested media.
- Locust bean gum (LBG) as a gelling agent for plant tissue culture mediaPublication . Gonçalves, Sandra; Romano, AnabelaLocust bean gum (LBG) is a natural hydrocolloid extracted from the seeds of carob tree (Ceratonia siliqua L.). This work describes the successful use of LBG as a gelling agent in combination with agar for shoot multiplication and rooting of carob tree and Iberian rose shoots. Its presence did not affect the multiplication rate of both species. The rooting frequency of carob shoots was even significantly increased in the presence of 5 g of LBG plus 4 g of agar to the medium compared to medium solidified with 9 g of agar. Iberian rose shoots rooting was not influenced by the addition of this gum to the rooting medium. Results obtained show that LBG can be used in combination with agar in culture medium as a gelling agent without negative effect on plant material and with the advantage of reduced medium costs.
- Headspace-SPME of in vitro shoot-cultures and micropropagated plants of Lavandula viridisPublication . Gonçalves, Sandra; Serra, Hugo; Nogueira, José Manuel F.; Romano, AnabelaIn this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L’Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7 - 79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.
- Carob trees (Ceratonia siliqua L.) regenerated in vitro can acclimatize successfully to match the field performance of seed-derived plantsPublication . Osório, Maria Leonor; Osório, Júlio; Gonçalves, Sandra; David, Maria Manuela; Romano, Anabela; Correia, Maria JoãoThe use of in vitro regenerated plants in forestry and orchard depends ultimately on the development of efficient transplantation protocols, ensuring high survival rates and successful establishment under field conditions. We tested the performance of micropropagated carob trees (Ceratonia siliqua L.) throughout the acclimatization process in terms of survival, growth and physiological traits, including field comparisons with seed-derived and mother plants. The field trial was 100 %successful, i.e. we found no major differences between micropropagated, seed-derived and mother plants in terms of growth rate, height, number of leaves, photosynthetic efficiency, chlorophyll fluorescence, chlorophyll content and soluble protein content, although these parameters changed significantly during acclimatization. Stomatal conductance (gs) was reduced by fourfold when plants were transferred from in vitro culture to the growth chamber, thus preventing uncontrolled wilting. The photosynthetic rate (PN) was relatively low in vitro, in the growth chamber and the greenhouse, but increased to match seed-derived and mother plants in the field. The chlorophyll a/b ratio in leaves from in vitro and growth chamber plants was typical of shade plants (2.1) but became more characteristic of sun plants in the subsequent acclimatization stages (3.1–3.5). The maximum efficiency of photosystem II (Fv/Fm) remained mostly constant at ~0.80 throughout acclimatization, as is typical for healthy, non-stressed plants. We conclude that our micropropagation and acclimatization protocols provide a suitable alternative to traditional mass propagation techniques for the carob tree.