Afonso, Clélia

Franco, Inês

Mouga, Teresa

English

This study is being financed by the project LIFE Berlengas - Conserving threatened habitats and species in Berlengas SPA through sustainable management, LIFE13 NAT/PT/000458.
To SPEA and ICNF a special thanks for all the help and support on the travels to the island and on the plants collection in the Natural Reserve of Berlenga Island.
This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE.info:eu-repo/semantics/publishedVersio

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ResultsSterilized by soaking in a bleach solution, and after that with ethanol (70%)Culture tubes with Murashige and Skoog (MS) mineral formulation (Sigma) supplemented with 0.3% (w/v) sucrose and 0.2% (w/v) gelrite(Sigma, Germany). Multiplication stage: shoots with 0.5-1cm high were placed in MS mineral salts, 3% (w/v) sucrose, 1ml.L-1Gamborg vitamin formulation (Sigma), 1.5% (w/v) Phytoagar and different growth regulatorsControl: Basal medium without the addition of any growth regulatorsThe Berlengas archipelago is located in the Atlantic Ocean, on thePortuguese continental shelf, on the western side of the IberianPeninsula, close to Cape Carvoeiro (Peniche). It distancesapproximately 5.7 miles from mainland. It is a protected area since1981.In 2011 Berlengas became part of the list of patrimony of UNESCO,aiming to conserve representative terrestrial and marine ecosystems ofthe Portuguese coast.As a result to insular isolation, and to very difficult climatic and edaphicconditions, terrestrial flora evolved differently from the species thatgrow in mainland, being smaller and more resistent.There are three endemic taxa, of great conservation value:•Armeria berlengensis,•Herniaria lusitanica subsp. berlengiana,•Pulicaria microcephala.•P. microcephala, as well as A. berlengensis, are registered in Annex II of theHabitats Directive, due to its conservation relevance, and are consideredvulnerable. Herniaria lusitanica subsp. berlengiana is registered as endangered.The aim of this project, therefore, was to multiply, in vitro, the species P. microcephala. Plant shoots were collected in the autumn 2015 from a natural wild population in the Berlengas’ Island.References[1] Gomes, C., Draper, D, Marques, I., Rosselló-Graell, A., 2004. Componente Vegetal do Plano de Ordenamento da Reserva Natural das Berlengas. Jardim Botânico.[2] Fay, M., 1992. Conservation of rare and endangered plants using in vitro methods. In Vitro Cell Dev Biol, 28P, 1-4. [3] Iriondo J., Pérez C, 1990. Application of in vitro culture techniques to the conservation of Iberian endemic endangered plant species. Botanic Gardens Micropropagation News, 1, 4–6.[4] Queiroga H., Leão F., Coutinho M. (coord), 2010, Nomination of the Berlengas Islands as a UNESCO Biosphere Reserve. Relatório para a Câmara Municipal de Peniche (Portugal).Acknowledgements This study is being financed by the project LIFE Berlengas - Conserving threatened habitats and species in Berlengas SPA through sustainable management, LIFE13 NAT/PT/000458. To SPEA and ICNF a special thanks for all the help and support on the travels to the island and on the plants collection in the Natural Reserve of Berlenga Island.This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE.Introduction Materials and methods ResultsDiscussions and ConclusionsIn vitro setting and multiplication of Pulicariamicrocephala, an endemic species from Berlengas Island Inês Franco1, Teresa Mouga2, Clélia Afonso21 ESTM, Instituto Politécnico de Leiria, 2520-641 Peniche, Portugal2 MARE – Marine and Environmental Sciences Centre, ESTM, Instituto Politécnico de Leiria, 2520-641 Peniche, PortugalCorresponding author e-mail: clelia@ipleiria.ptTable I. Concentration of the growth regulators of each medium, with the letter attributed to each combination.After two months of growth the plants demonstrate the impact of thepresence of growth regulators in the media.• Even in the presence of low concentrations of NAA and IBA (0.5mL/L, treatments A and S respectively) thenumber of root s is high.The control was the treatment that presents better results in terms ofabsence of vitrification and abnormal aspects.• Pulicaria microcephala, adapted to the Berlengas island’ conditions, didn’t performed well in in vitroconditions.The response to the growth hormones present in the media, excluding thecontrol situation, aren’t relevant.• The length of the shoots treated with Kin/NAA and KIN/IAA decreased with the increase of the hormonesconcentration. The higher the concentration of these hormones the higher the multiplication rates.• In the treatment with IBA, the impact of this hormone is more evident, as the number of shoots is higher.It is possible to multiply in vitro P. microcephala plantlets aiming toincrease the number of specimens on the Berlengas island.•More tests have to be performed in order to assess the success of theacclimatization and rooting stages.Regulatorconcentration(mL/L)NAA IAA IBA0,5 1 2 0,5 1 2 0,5 1 2Kin0,5 A B C J K L S T U1 D E F M N O V W X2 G H I P Q R Y Z ZZ0,0001,0002,0003,0004,0005,0006,0007,0001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28Mean Lenght (cm)Treatments:  A-I - KIN+NAA; J-R - KIN+IAA; S-ZZ - KIN+IBA±SD 0,0002,0004,0006,0008,00010,00012,00014,00016,00018,00020,0001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28MeannumberofbranchesTreatments:  A-I - KIN+NAA; J-R - KIN+IAA; S-ZZ - KIN+IBA±SD Figure 1. Preliminary results of in vitro shootmultiplication rates from P. microcephala: length ofthe highest shoot in response to various media. Theshoots were placed in medium with Kin in associationwith NAA (A-I), IAA (J-R) or IBA (S-ZZ). Letter codeaccording to the table I.Data represents mean ± Standard Deviation of 15replicates/treatment and were recorded after 4 weeksof culture.Figure 2. Preliminary results of in vitro shootmultiplication from P. microcephala: number of shootsthat formed during the assay in response to thepresence of various growth regulators in the media. Allof them with Kin in association with NAA (A-I), IAA (J-R)or IBA (S-ZZ). Variance also on the concentrationaccording to table I.Data represents mean ± Standard Deviation of 15replicates/treatment and was recorded after 8 weeksof culture.Figure 3. a) Expected look after four weeks in in vitro culture; b) Expected look after two months in in vitro culture; c) Typical resultafter one month in treatments with hormone regulators; d) Typical result after two months in treatments with hormone regulators; e)Examples of malformations, vitrified plants (1) and plants in rosettes (2); f) Callus formation ; g) In vitro flowering.(1)(2)(a) (c) (d)(b) (e) (f) (g)(a)(c) (d)(b)(a)(c) (d)(b)(a)(c) (d)(b)(a)(c) (d)(b)Figure 4. Examples of the look of the shoots under control treatment after a month (a) ortwo (c) in in vitro culture; Qualitative and comparative analysis of the plants roots subjectedto this treatment for one (b) or two (d) months respectively showed that plants almostreached the size of the culture flask in both times (a and c) but the roots did not showeddifferences between b) and d).Figure 4. Examples of the shoots under treatment A after a month (a) or two (c) in vitroculture; qualitative and comparative analysis of the plants’ roots subjected to thistreatment for one (b) or two (d) months respectively showed that plants are almost half ofthe size of the culture flask in both times (a and c) and the roots have elongated after twomonths (b and d). The formation of calli is evident in this photographs (b and d); Thistreatment has the lowest concentration of Kin and NAA growth hormones; The othersconcentrations show mostly the same results.Figure 5. Examples of the behavior of the shoots under treatment J after a month (a) or two(c) in in vitro culture; qualitative and comparative analysis of the plants’ roots subjected tothis treatment for one (b) or two (d) months respectively, showed that: the plants arealmost half to a third of the size of the culture flask in both times (a and c) and the rootshave elongated after two months (b and d). The formation of calli is evident in thesephotographs (b and d); this treatment is the one with the lowest concentration of Kin andIAA growth hormones; the others concentrations show almost the same results, for theroots; IAA causes the plant to present a lower number of roots, comparing to the control, Aor S treatments.Figure 6. Examples of the behavior of the shoots under treatment S after a month (a) or two(c) in in vitro culture; qualitative and comparative analysis of the plants roots subjected tothis treatment for one (b) or two (d) months respectively, showed that the plants are almosthalf to a third of the size of the culture flask in both times (a and c) and the roots haveelongated after two months (b and d). The formation of calli is also evident in thesephotographs (b and d); this treatment is the one with the lowest concentration of Kin andIBA growth hormones; IBA causes the plant to present a higher number of root, whencompared to the control, A or J treatments.

In-vitro setting and multiplication of Pulicaria microcephala, an endemic species from Berlengas Island

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In-vitro setting and multiplication of Pulicaria microcephala, an endemic species from Berlengas Island

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