Micropropagation of Helianthemum inaguae, a rare and endangered species from the Canary Islands

              Bot. Macaronesica 26: 55-64 (2006) 55
ISSN 0211-7150
MICROPROPAGATION OF HELIANTHEMUM INAGUAE, A RARE AND ENDANGERED SPECIES FROM THE CANARY ISLANDS
ISABEL SANTANA LÓPEZ1, FRANCISCO VALDÉS GONZÁLEZ2, & J. C. LUIS2
1 Jardín Botánico Canario “Viera y Clavijo”. Apto. 14 de Tafira Alta. C.P. 35017. Las Palmas de Gran Canaria, Gran Canaria, Canary Islands, Spain. E-mail: isantana@grancanaria.com.
2 Universidad de La Laguna, Facultad de Farmacia, GBVa (Grupo de Biología Vegetal Aplicada). Avenida Astrofísico Francisco Sánchez s/n, C.P. 38207. La Laguna, Tenerife, Canary Islands, Spain.
Recibido: Noviembre 2005
Key words: Cistaceae, endangered species, Helianthemum inaguae, micropropagation, shoot multiplication, rooting
Abbreviations: BA–Benzyladenine, Kin–kinetin, IBA–indolebutiric acid, MS–Murashige and Skoog media, NAA–naphthalenacetic acid.
Palabras clave: Cistaceae, especies amenazadas, Helianthemum inaguae, micropropagación, multiplicación por segmentos nodales, enraizamiento.
Abreviaciones: BA–Benciladenine, Kin–kinetina, IBA– Ácido Indolbutírico, MS– medio Murashige y Skoog, NAA– Ácido naftalenacético.
SUMMARY
A micropropagation protocol has been developed for Helianthemum inaguae Marrero Rodr., González Martín & González Artiles, an endangered species from Gran Canaria (Canary Islands, Spain). Shoot tips and nodal segments isolated from in vitro germinated seedlings, aged two months, were used as primary explants. Multiple shoot production was obtained using MS medium supplemented with different concentrations of BA, Kin and NAA, showing BA and Kin the best results. Finally, rooting was achieved also in MS medium by adding IBA or without any plant growth regulator. A 72% survival rate was obtained during the acclimatisation process.
RESUMEN
El presente trabajo da a conocer un protocolo de micropropagación desarrollado para Helianthemum inaguae Marrero Rodr., González Martín & González Artiles, una de las especies más amenazadas de la flora endémica de Gran Canaria (Islas Canarias, España). Como explantos iniciales se utilizaron yemas apicales y segmentos nodales aislados de semillas germinadas in vitro de unos dos meses de edad. La multiplicación de yemas se realizó en medio MS al que se añadieron diferentes concentraciones de BA, Kin y NAA, observándose los mejores resultados con BA y Kin. Finalmente, el enraizamiento se llevó a cabo en medio MS sin reguladores o con la adición de diferentes concentraciones de IBA. Durante el proceso de aclimatación a las condiciones ex vitro se obtuvo un 72 % de supervivencia. 56 ISABEL SANTANA LÓPEZ, FRANCISCO VALDÉS GONZÁLEZ, & J. C. LUIS
INTRODUCTION
The genus Helianthemum in the Canary Islands is characterised, firstly, for the local distribution of their species and, secondly, for the small number of individuals in their natural populations (SVENTENIUS, 1960; BRAMWELL et al., 1977; MARRERO, 1992; MARRERO et al, 1995) which causes a constant loss of alleles that together with the extinction of their habitats could reduce the ability of Helianthemum species to increase in number of individuals, to evolve, and ultimately to adapt to future changing circumstances.
Since Helianthemum inaguae was described (MARRERO, 1995, op. cit.), only one population has been located in La Montaña de los Hornos (Inagua) in the South West of Gran Canaria and, therefore, the H. inaguae is considered “in danger of extinction” and included in legal catalogues of threatened plants (CNEA: Catálogo Nacional de Especies Amenazadas (1998) and CEAC: Catálogo de Especies Amenazadas de Canarias (2001)) for its preservation. Moreover, from its last conservation status review (MARRERO et al., in BAÑARES et al. 2003), according with the IUCN criteria (IUCN, 2001), H. inaguae is also included in the Critically Endangered (CR) list.
In this paper we present a reliable protocol for the micropropagation of H. inaguae, in order to be able to increase the number of individuals in the natural populations. Seeds from the collection of Jardín Botánico Canario “Viera y Clavijo”, were used as a starting plant material in the micropropagation protocol, a fact that reduces over-collecting the natural population of H. inaguae.
MATERIAL AND METHODS
2.1. Plant Material
Seeds of Helianthemum inaguae were obtained from the collection of the Jardín Botánico Canario “Viera y Clavijo”, which were conserved in the institution since 2002.
2.2. Sterilisation Protocol
Seeds were washed in running water and Tween 20 for several minutes and then immersed in ethanol (70%) for 30 seconds. Following this treatment, the seeds were sterilised in two steps with Tween 20 and commercial bleach at two different concentrations (2 g l-1 of active chlorine the first one and 1 g l-1 the second). Finally, all the seeds were washed three times with distilled sterile water before been used in the germination tests.
2.3. Germination Tests
Germination was carried out in Petri dishes with 20 ml of MS medium supplemented with 30 g l-1 of sucrose and 4.5 g l-1 of agar. Incubation was carried out at 20 ºC under photoperiod conditions (16 hours light / 8 hours dark). Seeds were soaked in distilled water during 24 hours or scarified with sulphuric acid (98%) during 2 minutes to, later, combine both treatments in order to test the germination ability of H. inaguae. Radicle emergence was the criterion for deciding MICROPROPAGATION OF HELIANTHEMUM INAGUAE, A RARE AND ENDANGERED SPECIES 57
whether germination had taken place. The amount of germinated seeds was registered every seven days for a period of 4 weeks.
2.4. Micropropagation, Rooting and Acclimatisation
After 30 days, plants from germinated seeds were placed in tubes with 20 ml of basal MS medium. Shoot tips and nodal segments were propagated on MS medium with several concentrations of BA, Kin and NAA (Table 1). One centimetre or longer healthy shoots were rooted by using MS medium with different concentrations of IBA (1, 2, 3, 4 mg l-1) or without plant growth regulator. All plant material were maintained at 20 ºC with 16/8 hours of photoperiod and subcultured after 30 ± 5 days.
Only healthy well-developed and rooted plants were used for the acclimatisation in the greenhouse. They were previously washed in sterile water to eliminate the excess of agar and then treated with a fungicide solution (Carbendacima, 1 g l-1), to be potted into 7.5 cm diameter pots filled with a sterilised soil: peat: vermiculite mix (2:1:1). Finally, they were introduced in trays and covered with transparent plastic. During acclimatisation, aeration was progressively increased by the partial removal of the plastic cover.
All the experiments were repeated at least three times always obtaining similar results. The values represent the mean plus the standard deviation. During the micropropagation, differences between treatments were analysed comparing the means by using the Mann- Whitney test. Significant results were obtained for p< 0.05.
PLANT GROWTH REGULATOR
MEDIUM
BA
Kin
NAA
0
-
-
-
1
0
-
-
2
0.5
-
-
3
1
-
-
4
1.5
-
-
5
2
-
-
6
-
0
-
7
-
0.5
-
8
-
1
-
9
-
1.5
-
10
-
2
-
11
1
-
0.1
12
1
-
0.1
13
-
1
0.1
14
-
1
0.1
Table 1. Plant growth regulators and concentrations used in mg l-1 for the micropropagation of H. inaguae. 58 ISABEL SANTANA LÓPEZ, FRANCISCO VALDÉS GONZÁLEZ, & J. C. LUIS
RESULTS AND DISCUSSION
3.1. In Vitro Germination of Helianthemum inaguae Seeds
The germination of Helianthemum inaguae seeds was successfully observed in all the treatments used, although significant differences were obtained. Soaking the seeds in distilled water during 24 hours combined with a scarification in H2SO4 (98%) during 2 minutes significantly stimulated seeds radicle emergence, reaching an 80% of germination in one week. All other treatments showed a lower germination rate (below 50%) needing much longer time, two and four weeks (Figure 1).
Figure 1. Percentage of germinated seeds (a) and the number of weeks needed to reach the percentage of germination (b) for the four different treatments used. T1: no treatment; T2: soaking in distilled water during 24 hours; T3: scarification in H2SO4 during 2 minutes; T4: soaking in distilled water during 24 hours and scarification in H2SO4 during 2 minutes.
These germination results corroborate previous studies with other species of Helianthemum (SANTANA-LÓPEZ et al., 2004; IRIONDO et al., 1995, CORRAL et al., 1990), showing the scarification as a necessary pre-treatment to improve seeds germination. However, in this study the scarification process followed by the sterilisation with commercial bleach inhibited, in 15% of the cases, the development of the germinated seeds, producing necrotic areas in their primary leaves. Nevertheless, despite this problem, combining scarification and soaking during 24 hours was the best treatment to stimulate the germination of H. inaguae seeds.MICROPROPAGATION OF HELIANTHEMUM INAGUAE, A RARE AND ENDANGERED SPECIES 59
3.2. Micropropagation of Helianthemum inaguae.
In vitro micropropagation of H. inaguae was studied by using MS medium with different concentrations of BA, Kin and NAA. Best results were obtained with BA at 1 and 1.5 mg l-1, observing higher shoots length only when 1 mg l-1 was used. Both, lower or higher concentrations of BA, such as 0.5 mg l-1 or 2 mg l-1, showed lower production of new shoots per explant; however, higher shoot length was observed with 0.5 mg l-1 of BA (Figure 2).
Figure 2. Number of shoots per explant (a) and length of the shoots in cm (b) when different concentrations of BA were used. The values represent the mean plus the standard deviation. Significant results were obtained for p< 0.05 and represented with different letters over the bars.
When Kin was present in the medium, almost all concentrations exhibited similar shoots production per explant but significantly lower if compared with BA. However, in contrast with BA, the length of the shoots was well over 1 cm in three of the Kin concentrations tested (Figure 3). These results corroborate previous studies where BA stimulates multiple shoot formation while Kin is more efficient in the elongation process (SANTANA-LÓPEZ et al., 2004; IRIONDO et al., 1995, MORTE and HONRUBIA, 1992). Furthermore, we used low concentrations of Kin (0.2 mg l-1) to stimulate shoots elongation after a BA treatment, always obtaining good results (data not shown). Finally, the presence of NAA in the propagation medium, either with BA or Kin, induced callus formation with no morphogenetic structures in a high proportion of the explants. 60 ISABEL SANTANA LÓPEZ, FRANCISCO VALDÉS GONZÁLEZ, & J. C. LUIS
Figure 3. Number of shoots per explant (a) and length of the shoots in cm (b) when different concentrations of Kin were used. The values represent the mean plus their standard deviation. Significant results were obtained for p< 0.05 and represented with different letters over the bars.
On the other hand, during the multiplication studies with BA hyperhydric tissues were observed in the explants. In fact, when using 1 mg l-1or higher concentrations of BA almost 50% of the plant material showed hyperhydric characteristics. However, this trend was not observed when Kin was used (Figure 4). Further studies are needed in order to be able to minimise this effect.
Figure 4. Percentage of hyperhydric tissues when different concentrations of BA and Kin were used. MICROPROPAGATION OF HELIANTHEMUM INAGUAE, A RARE AND ENDANGERED SPECIES 61
Figure 5. Hyperhydric tissues in Helianthemum inaguae plants during the multiplication process when BA and KIN were present in the media (a) BA 0,5 mg L-1 (b) BA 1 mg L-1; (c) Kin 0,5 mg L-1 (d) Kin 1 mg L-1. Black arrows indicate plant tissues with hyperhydric simptoms. 62 ISABEL SANTANA LÓPEZ, FRANCISCO VALDÉS GONZÁLEZ, & J. C. LUIS
3.3. Rooting and Acclimatisation
Rooting in vitro Helianthemum inaguae plants was successful either with different IBA concentrations or without any plant growth regulator. However, significant differences were observed between IBA treatments concerning the number and the length of developed roots (Figure 6). A higher number of roots were observed when using higher concentrations of IBA, however, a higher length was observed when lower concentrations of IBA were used. In addition, higher concentrations of IBA (3 and 4 mg l-1) stimulated the formation of callus, which could have a negative effect in their acclimatisation process due to the formation of non-functional roots (Figure 7). Similar results were obtained for other Helianthemum species (SANTANA-LÓPEZ, et al., 2004; IRIONDO et al. 1995).
For their acclimatisation, well-rooted and healthy plants were transferred to the greenhouse and later to an appropriate area in the Jardín Botánico “Viera y Clavijo”, where conditions were similar to their natural habitat. A 72% survival rate was observed after the acclimatisation process.
CONCLUSIONS
In summary, the results of this study showed that the protocol developed is an efficient method for the micropropagation of Helianthemum inaguae. This method allowed us to start with a relative small amount of plant material reaching at the end of the acclimatisation process a 72% plant survival, thus contributing to the species preservation. Despite these positive results some aspects, such as the development of hyperhydric tissues during the multiplication with BA, should be studied in detail in a near future.
ACKNOWLEDGEMENTS
We want to thank the Cabildo of Gran Canaria for the post-graduate studentship given to the Jardín Botánico “Viera y Clavijo”, which made this work possible. We are also grateful to David Bramwell and José Naranjo Suárez for their support throughout the process and to Blas Vilches Navarrete, Alicia Roca Salinas, Margot Ravelo Armas and Emma Suárez Toste for their collaboration in this work.
REFERENCES
ANÓNIMO, 1989.- Ley 4/1989, de 27 de marzo, de Conservación de los Espacios Naturales y de la Flora y de la Fauna Silvestre (B.O.E. de 28 de marzo de 1989, nº 74).
ANÓNIMO, 1998.- Orden de 9 de julio de 1998 por la que se incluyen determinadas especies en el Catálogo Nacional de Especies Amenazadas y cambian de categoría otras especies que ya están incluidas en el mismo (B.O.E. de 20 de julio de 1998, nº 172).
ANÓNIMO, 2001.- Decreto 151/2001, de 23 de julio, por el que se crea el Catálogo de Especies Amenazadas de Canarias (B.O.C. de 1 de agosto de 2001, nº 97)
BAÑARES, Á., G. BLANCA, J. GÜEMES, J.C. MORENO, & S. ORTIZ, eds., 2003.- Atlas y Libro Rojo de la Flora Vascular Amenazada de España. Dirección General de Conservación de la Naturaleza. Madrid, 1072 pp. MICROPROPAGATION OF HELIANTHEMUM INAGUAE, A RARE AND ENDANGERED SPECIES 63
Figure 6. Number of roots per explant (a) and length of the roots in cm (b) when different concentrations of IBA were used. The values represent the mean plus the standard deviation. Significant results were obtained for p< 0.05 and represented with different letters over the bars.
Figure 7. Callus formation in H. inaguae plants with 3 mg L-1 of IBA (a) and well rooted plants with 0,5 g L-1 of IBa (b). 64 ISABEL SANTANA LÓPEZ, FRANCISCO VALDÉS GONZÁLEZ, & J. C. LUIS
BRAMWELL, D., J. ORTEGA, & B. NAVARRO, 1977. – Helianthemum tholiforme a new species of Cistaceae from Gran Canaria. Botánica Macaronésica 2 (1976): 69 – 74.
CORRAL, R., PITA J. M. & PÉREZ-GARCÍA, F., 1990.- Some aspects of seed germination in four species of Cistus L. Seed Sci. & Technol., 18, 321-325.
IRIONDO, J. M., C. PRIETO, AND F. PÉREZ-GARCÍA. 1995 In vitro regeneration of Helianthemum polygonoides Peinado et al., an endangered salt meadow species. Botanical Gardens Micropropagation News, Vol 2 Part 1.
IUCN, 2001. - IUCN Red List Categories and Criteria: Version 3.1. IUCN Species survival Commission. IUCN, Gland, Switzerland and Cambridge, UK. ii + 30 pp.
MARRERO RODRÍGUEZ A., M. GONZÁLEZ MARTÍN & F. GONZÁLEZ ARTILES, 1995. - Descripción de una nueva especie de Helianthemum Miller para Gran Canaria, Islas Canarias. Botánica Macaronásica 22: 3 – 12.
MARRERO, Á., 1992. – Notas taxonómicas del género Helianthemum Miller en Lanzarote. Botánica Macaronésica 19/20: 65 – 78.
MORTE, M. A. & M. HONRUBIA. 1992. - In vitro propagation of Helianthemum almeriense Pau (Cistaceae). Agronomie 12, 807-809.
SANTANA LÓPEZ, I., J. C. LUIS, M. RAVELO ARMAS & F. VALDÉS GONZÁLEZ. 2004. - In vitro propagation of Helianthemum bystropogophyllum Svent., a rare and endangered species from Gran Canaria (Canary Islands). Botánica Macaronésica 25: 71-77.
SVENTENIUS, E. S. 1960. – Additamentum ad floram canariensem 1. Madrid: Inst. Nac. Invest. Agron. – 93 pp. il.