Volume 1 Part 8 March 1995

Explant establishment in the micropropagation of Globularia ascanii, a threatened species from Gran Canaria

Miguel A. Cabrera-Pérez, Viceconsejería de Medio Ambiente, Vivero Forestal de Tafira, Carretera del Centro Km 6.4, CP 35017 Las Palmas de Gran Canaria, Spain

Abstract

Globularia ascanii Bramw. & Kunk. is an endangered endemic from Gran Canaria. Different explants were used to initiate cultures for shoot bud formation. Apical buds and nodal segments of adult plants showed a high percentage of contamination and lethal browning. The use of explants from aseptically germinated seedlings to establish in vitro cultures was effective in overcoming contamination and problems associated with exudates. Other in vitro techniques for controlling browning are discussed.

Introduction

The genus Globularia consists of about 30 species in the Mediterranean region, and its southern limit is in Macaronesia (Bramwell & Kunkel, 1974). There are three species in the Canary Islands: G. salicina Lam., G. sarcophylla Svent. and G. ascanii Bramw. & Kunk. The distribution of G. ascanii is restricted to a very small region in the north west of Gran Canaria (Tamadaba Massif) and a only few small populations have been found. It is an endangered species (E according to the IUCN Red Data Book categories: Barreno-Rodríguez et al., 1983). However, few reports of propagation of this species exist. Maya et al. (1988) failed to achieve germination of achenes from cultivated plants. On the other hand, although the percentage of fruits containing seeds was low, González-Martín et al. (1994) achieved high percentage germination using seeds from wild plants and different incubation conditions.

In vitro culture techniques can rapidly increase the number of individuals of endangered species with reproductive problems and/or extremely reduced populations (Iriondo & Pérez, 1990). Thus these techniques could be useful in the conservation of G. ascanii.

Lethal browning occurs in some plant tissue cultures, especially woody plants, and is generally considered to result from the oxidation of phenolic compounds released from the cut ends of the explants (Bhat & Chandel, 1991). The oxidized products, e.g. quinones, inhibit enzyme activity, leading to the death of explants (Hu & Wang, 1983). Previous work with G. sarcophylla at Kew showed that explant establishment was problematic as a result of contamination and necrosis (Synge, 1991). The most effective method of preventing browning was regular transfer every 6-8 weeks to Murashige & Skoog (1962) medium (MS) + 2 g/l charcoal, but without growth regulators (Wilkinson, pers. comm.). Plants were, however, regenerated from these cultures (Fay, 1993). These problems were also seen in preliminary trials with cultures of G. ascanii. A first attempt to overcome the harmful effects of browning and to develop an effective method for micropropagation of G. ascanii is described.

Materials and Methods

Apical buds and nodal segments of adult plants were surface sterilized using different treatments. All explants were soaked in 70% ethanol for 1 min. Apical buds were sterilized in 1% (v/v) commercial bleach (4% NaOCl) for 5 min (T-1) or 15 min (T-2), while nodal segments were soaked in 1% (T-3) or 5% (T-4) commercial bleach solution for 15 min. The explants were rinsed three times with autoclaved distilled water, and were placed on MS with different combinations of benzylaminopurine (BAP) and naphthaleneacetic acid (NAA). Cultures were maintained at 25±2°C with a 16h photoperiod.

Seeds from wild plants were surface disinfected in 70% ethanol for 1 min, followed by 5 min in 1% (w/v) calcium hypochlorite and washed three times in distilled water. Seeds were inoculated on MS without growth regulators and were incubated at 20±1°C with a 16h photoperiod. Apical and nodal segments excised from germinated seeds after six months growth, were placed on MS + 4.44 µM BAP and 0.54 µM NAA. Cultures were maintained at 20±1°C with a 16 h photoperiod.

All media were supplemented with 3% sucrose and 0.8% agar and were adjusted to pH 5.8 with KOH. There were 12-24 explants in each replicate and at least two replicates/experiment.

Results and Discussion

In all treatments, apical buds and nodal segments from adult plants showed a high percentage of contamination. At least 80% of nodal segments were contaminated, whereas contamination rates for apical buds were lower (Figure 1). The increase in sterilization time for apical buds or NaOCl concentration for nodal segments reduced contamination. Lethal browning occurred at the cut surface of all explants after a few days, and later developed throughout the explant and culture medium. These problems meant that the influence of growth regulators on bud formation could not be assessed.

Figure 1. Percentage of contamined explants of G. ascanii after 20 days of culture: a) nodal segments; b) apical buds.


Germination percentage was lower than that obtained by González-Martín et al. (1994), but only one culture was contaminated after six months of culture. Apical and nodal segments excised from germinated seeds showed slight browning at the cut ends and in the culture medium a few days after inoculation, but it was only lethal for some more basal explants. After a few weeks of culture, the development of apical buds and the formation of lateral buds was observed on MS medium + 4.44 µM BAP and 0.54 µM NAA.

Choice of juvenile explants has been shown to reduce contamination rate (Haldeman et al., 1987) and browning (Bhat & Chandel, 1991). In this study, use of young plant material showed the potential for increasing the quantity of explants for micropropagation of G. ascanii, due to the reduction in contamination and lethal browning. The quantity of explants might have been higher if we had used combinations of other traditional methods for reducing exudates. Soaking explants in solutions containing antioxidants prior to inoculation and/or the addition of antioxidants to culture media can be useful for preventing polymerization of quinones and reducing the probability that they will react with protein (Compton & Preece, 1986). Novel techniques e.g. sealing the cut ends with paraffin wax for prevent exudation (Bhat & Chandel, 1991) could also be used. Success in reducing the impact of these problems would help the study of the effect of growth regulators and establish an effective method for the micropropagation and conservation of G. ascanii.

Acknowledgements

I thank Dr. Francisco Valdés of La Laguna University for his comments and suggestions and the Dpto. de Biología of Las Palmas University for facilitating my work.

References

Barreno-Rodríguez et al. (1983). Lista de las plantas endémicas, raras o amenazadas de España. Información Ambiental 3: 48-71.

Bhat S.R. & Chandel K.P.S. (1991) A novel technique to overcome browning in tissue culture. Plant Cell Rep. 10:358-361.

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Compton M.E. & Preece J.E. (1986) Exudation and explant establishment. I.A.P.T.C. Newsletter 50:9-18.

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Hu C.Y. & Wang P.J. (1983) Meristem shoot tip and bud cultures. In: D.A. Evans, W.R. Sharp, P.V. Ammirato, Y. Yamada (eds.). Handbook of Plant Cell Cultures, Vol 1. MacMillan, New York. 177-217.

Iriondo J.M. and Pérez C. (1990) Micropropagation of an endangered species: Coronopus navassi (Brassicaceae). Plant Cell Rep. 8:745-748.

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