Volume 2 Part 2 July 1996
M.D. Lledó 1, M.B. Crespo 1 & J.B. Amo-Marco 2
1 Departamento de Ciencias Ambientales y Recursos Naturales (Botánica), Universidad de Alicante, PO Box 99. 03080 Alicante, Spain
2 Departamento de Biología Vegetal, Universitat de València. C/ Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain
Limonium thiniense Erben is a threatened plant endemic to a few localities in southeastern Spain. Seeds from a herbarium sheet were used for initiating aseptic cultures. Several cytokinins (2iP, BA, and Kin) were tested in the multiplication phase with MS medium. Auxins (IAA, IBA, and NAA) were also used for rooting on half MS. Regenerated plants are successfully growing and flowering at the University of Alicante.
Limonium Miller (Plumbaginaceae) consists mainly of rosulate plants with showy inflorescences found on maritime cliffs or saline soils. Due to the natural spatial fragmentation of these ecosystems, small populations of this genus are in active speciation, and populations with particular characteristics (microspecies and microendemics) can be found in small coastal areas or salt marshes.
The reproductive systems of these plants facilitate the occurance of hybrids between microendemics and widespread species with larger distributions. Therefore the utilisation of seed for conservation purposes should be avoided in many cases (Lledó et al., 1993). Nevertheless, seed as well as axillary buds from adult plants (Fay, 1993) have been used as source material for conserving threatened Limonium species (Martín & Pérez, 1992; Martín & Pérez, 1995; Fay, 1994). Therefore the appropriate source of plant material should be assessed for each endemic.
L. thiniense was described in 1981 from materials collected in a salt marsh near Santa Pola (Alicante, SE Spain), where it occured with several Limonium spp, many of them endemics to eastern Spain: L. santapolense Erben, L. parvibracteatum Pignatti, L. bellidifolium (Gouan) Dumort., L. delicatulum (Girard) O. Kuntze, L. cossonianum O. Kuntze, L. angustebracteatum Erben, L. caesium (Girard) O. Kuntze, L. echioides (L.) Mill., L. furfuraceum (Lag.) O. Kuntze, etc. No hybrids between L. thiniense and other species have been reported. In fact, the only closely related species with which it could possibly hybridize (L. cavanillesii Erben) is endemic to the Castellón coast, more than 250 km north of Alicante.
In 1992, an ex situ conservation plan involving in vitro cultures was initiated, but unfortunately it was impossible to find the only known population in Santa Pola. The only living material available was a few seeds obtained from herbarium specimens collected in Santa Pola at the end of 1990, and conserved at the Herbarium of the University of Alicante (ABH 1740). Although seed dormancy problems have never been reported in this group, seeds of L. thiniense were difficult to germinate, and a specific methodology for germinating them was developed for initiation of aseptic cultures.
Two different factors were taken into account for developing a method to germinate seeds: i) the effect of GA3 (gibberellic acid) and ii) photoperiod.
Seeds of L. thiniense were soaked for 24 hours in GA3 (0, 50, and 400 ppm). They were then surface sterilised with ethanol (70%) for 2 min and calcium hypochlorite (6% w/v) for 30 min and rinsed four times in sterile distilled water. Seeds were transferred to hormone-free half strength MS (MS/2; Murashige & Skoog, 1962) in Petri dishes. Cultures were grown at 23°C, half in a 16 h photoperiod with 60 µM m-2s-1 and the rest in the dark. After one week, the seedlings were counted.
After two weeks, those seedlings germinated in light with 400 ppm GA3 were used for initiating the multiplication process. Root parts were removed, and both hypo- and epicotyls were transferred to flasks containing 25 ml of MS supplemented with 2mg/l Kinetin (K). Every two months, they were subcultured onto fresh medium.
For studying both multiplication and rooting stages, small rosettes with 3-5 leaves were used as single explants in subsequent experiments.
MS + K, benzyladenine (BA) or 2-isopentenyladenine (2iP) (0, 0·1, 0·5, 1, 2 or 5 mg/l) was used for testing multiplication rate. For rooting, MS/2 medium + indoleacetic acid (IAA), indolebutyric acid (IBA) or naphthylacetic acid (NAA) (0 or 0·5 mg/l) was used. All media were supplemented with MS vitamins, sucrose (20 g/l), and Probus agar (0·7%). Before autoclaving (20 min, 121°C) pH was adjusted to 5·7-5·8 with KOH. Cultures were grown at 23°C under a 16 h photoperiod (60 µM m-2s-1).
Rooted plantlets were transplanted into 5 cm pots containing equal proportions of peat moss and vermiculite. Plants were then acclimatised gradually to field conditions from the greenhouse.
Since the herbarium sheet of L. thiniense had been disinfested by freezing, seeds were difficult to germinate. However, after imbibition in a non-physiological concentration of GA3 (400 ppm) high percentage germination was obtained (Table 1). No germination was achieved with lower concentrations, probably due to the hard testa resticting uptake of the regulator.
Table 1. Effect of GA3 and light on germination of Limonium thiniense seeds
Photoperiod GA3 (mg/l) Germination (%)
16 h light 0 0 24 h dark 0 0 16 h light 50 0 24 h dark 50 25 16 h light 400 67 24 h dark 400 85
Germination percentages might have been increased by softening the testa by scarification with sand or emery paper, or by chemical treatments with strong acids, but these treatments could have damaged the embryo and reduced the viability of plantlets. Therefore they were not used to avoid unnecessary losses of the scant material.
Plantlets grown in the light after imbibition (400 ppm GA3 ) were used for initiating aseptic cultures on MS medium + 2 mg/l Kin. Although the percentage of seeds which germinated in the dark was higher, they presented problems when transferred into light.
Cultures initiated from that material presented a similar response to cultures obtained from adult material. Since the multiplication rate was lower than in other Limonium spp., after 4 subcultures enough small rosettes had been formed to start multiplication and rooting tests.
Multiplication rate was significantly higher in cultures supplemented with low concentrations of BA (0.1-0.5 mg/l) or higher concentrations of K (1-2 mg/l) or 2iP (5 mg/l) (Table 2), but with 2iP callus formation was detected. Although the multiplication rate in L. thiniense was lower than in other Limonium spp., the high viability of cultures ensured the conservation of this endemic species.
Table 2. Effect of K, BA, 2iP on the number of rosettes produced per cluster, viability (V), and percentage of callus formation in in vitro cultures of Limonium thiniense. Different letters indicate significantly different means (Tukey Test P< 0.05).
Cytokinin number of buds ± V (%) Callus (mg/l) S.E. (%) hormone-free 1.0 ± 0.0 d 100 20 K 0.1 1.0 ± 0.0 d 100 10 0.5 1.9 ± 0.4 bcd 100 10 1 2.4 ± 0.5 abcd 100 20 2 4.6 ± 0.8 ab 100 20 5 2.1 ± 0.7 bcd 100 0 BA 0.1 3.0 ± 1.5 abcd 100 20 0.5 3.8 ± 1.7 abcd 100 0 1 5.0 ± 0.7 a 100 40 2 1.4 ± 0.2 cd 100 40 5 2.2 ± 0.7 bcd 100 30 2iP 0.1 1.0 ± 0.0 d 100 55 0.5 1.8 ± 0.5 bcd 90 45 1 1.2 ± 0.2 cd 100 40 2 1.8 ± 0.3 cd 100 85 5 4.1 ± 0.8 abc 100 60
For other endemic Limonium spp., the use of auxins together with BA in the multiplication stage induced the formation of many small buds (Martín & Pérez, 1992), and an additional elongation stage was needed before rooting. In our case, a simplification of the process was preferred to the production of many clones of the same plant, that would reduce the level of biodiversity being conserved.
During the rooting stage, the response was similar with hormone-free medium, and with media containing IAA or IBA (Table 3). NAA promoted callus formation, and this material was not useful for acclimatisation. Rooted plants produced on other media are successfully growing in vivo at the University of Alicante, and flowered normally in June to August 1995. No phenotypic variations were detected between the micropropagated plants and natural populations.
Table 3. Effect of several rooting media on root and callus formation, and on root length in in vitro cultures of Limonium thiniense.
Salt Auxin rooting root length mm Callus medium (mg/l) (%) ± S.E. (%) MS/2 -- 90 31.7 ± 6.1 0 MS/2 0.5 IAA 100 29.4 ± 3.9 100 MS/2 0.5 IBA 90 21.8 ± 4.1 70 MS/2 0.5 NAA 40 12.7 ± 2.2 100
In the last three years, new populations of L. thiniense have been found in the southeastern Iberian Peninsula, specifically in Murcia and Albacete Provinces (Erben, 1993), and four additional localities have recently been discovered in Alicante Province (Aguilella et al., 1994). Thus, the current situation of the species is not as critical as in 1992, and now this plant should be given the Vulnerable category of threat (IUCN).
The micropropagation of this plant was not only an investigation of an ex situ conservation method, but also of the utility of herbarium materials for in vitro techniques. In the last decade herbaria have become an increasingly important tool for biodiversity studies. They are in fact repositories not only of dried specimens for morphological studies, but can also be a source of DNA for phylogenetics (Cano & Poinar 1993, Savolainen et al., 1995), and even of living material which can be used to conserve endangered plants as described here.
Aguilella A., Carretero J.L., Crespo M.B., Figuerola R. & Mateo G. (1994) Flora vascular rara, endémica o amenazada de la Comunidad Valenciana. Generalitat Valenciana, Valencia.
Cano R.J. & Poinar H.N. (1993) Rapid isolation of DNA from fossil and museum specimens suitable for PCR. Biotechniques 15: 432-435.
Erben M. (1993) Limonium Miller. In: Castroviejo & et al. (eds.), Flora Iberica 3. Real Jardín Botánico, CSIC. Madrid.
Fay M.F. (1993) Micropropagation of plants from the Canary Islands. Bol. Mus. Mun. Funchal. 2: 85-88.
Fay M.F. (1994) In what situation is in vitro culture appropriate to plant conservation? Biodiv. Conserv. 3: 176-183
Lledó M.D., Crespo M.B. & Amo-Marco J.B. (1993) Preliminary remarks on micropropagation of threatened Limonium species (Plumbaginaceae). Bot. Gard. Microprop. News. 1: 72-74.
Martín C. & Pérez C. (1995) Micropropagation of five endemic species of Limonium from the Iberian Peninsula. J. Hort. Sci. 70(1): 97-103.
Martín C. & Pérez C. (1992) Multiplication in vitro of Limonium estevei Fdez. Casas. Ann. Bot. 70: 165-167.
Murashige, T. & Skoog, F. (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.
Savolainen V., Cuénoud Ph., Spichiger R., Martínez M.D.P., Crèvecoeur M. & Manen J.F. (1995) The use of herbarium specimens in DNA phylogenetics: evaluation and improvement. Pl. Syst. Evol. 197:87-98
This work was supported by an F.P.I. grant from Conselleria d'Educació i Ciència (Generalitat Valenciana) to M.D. Lledó, and by the Investigation Agreement GV-0861/93 between Conselleria de Medi Ambient (Generalitat Valenciana) and Universitat de València.