Volume 2 Part 1 September 1995

In vitro regeneration of Helianthemum polygonoides Peinado et al., an endangered salt meadow species

J.M. Iriondo, C. Prieto and F. Pérez-García
Dpto. Biología Vegetal, E.U.I.T. Agrícola Universidad Politécnica, E-28040 Madrid, Spain


A method for micropropagation of Helianthemum polygonoides , a narrow endemic of salt meadows in SE Spain has been developed. Primary explants were shoot tips from plantlets obtained from in vitro germinated seeds. The highest shoot proliferation rates were obtained on MS + 1 mg/l BA + 0.1 mg/l NAA. Rooting occurred on MS medium alone or with varying concentrations of IBA. The highest number of roots per explant was obtained on MS + 4 mg/l IBA. An 87% survival rate was achieved when the plantlets were transferred to the greenhouse.


BA - benzyladenine, Kin - kinetin, GA3 - gibberellic acid, NAA - naphthalenacetic acid, IBA - indole-butyric acid, MS - Murashige & Skoog.


Helianthemum polygonoides Peinado et al. (Cistaceae) is a Spanish endemic with a very narrow distribution (Peinado et al., 1987). It is only found in a 24 km2 area around Tobarra (Albacete, Spain). This perennial chamaephyte is adapted to a very specific habitat, salt meadows located in depressions filled with salts rich slime deposits (López-González, 1993).

As with other members of Cistaceae currently used in horticulture, its yellow flowers, although very delicate, are produced in great abundance and are notable for their size and singular beauty.

Considering the narrow distribution of the species and the susceptibility of its natural habitat, H. polygonoides is classified as vulnerable (V) according to the IUCN categories. The status of the species and the existence of specific threats, such as the establishment of a water treatment plant at one of the populations, caused us to adopt ex situ conservation measures via the preservation of a representative sample of seeds at the Germplasm Bank of the Departamento de Biología Vegetal, Universidad Politécnica de Madrid, and the development of micropropagation techniques.

In vitro culture techniques are widely used in plant conservation with a large number of species (Fay, 1994). Nevertheless, previous literature on the in vitro culture of Cistaceae is scarce. We only have knowledge of two works, one on the micropropagation of Cistus x purpureus (M'Kada et al. , 1991) and the other on Helianthemum almeriense (Morte & Honrubia, 1992).

The aim of this work was to develop a method for the micropropagation of Helianthemum polygonoides.

Materials and methods

Seeds of Helianthemum polygonoides were collected in 1993 at the Cordovilla salt meadow (Tobarra, Spain). The seeds were desiccated to 4% water content and stored at -10o C in the seed bank.

In September 1993, 300 seeds were taken to the laboratory, pretreated by immersion in 7O% (v/v) ethanol for 1 min., surface disinfected with 0.5% NaOCl for 20 min. and rinsed three times in sterile distilled water at 90 oC. The seeds were left in the last water bath for 24 h while the water cooled down. Seeds were then aseptically transferred to 8 x 11 cm glass containers with 30 ml MS (Murashige & Skoog, 1962) medium + 3% sucrose. All medium constituents were added before adjusting the pH to 5.8. After adding 0.8% Difco Bacto agar, media were autoclaved at 121oC for 20 min. and then dispensed into containers.

Sixty days later, shoot tip explants about 10 mm long were excised from the seedlings and transferred to MS medium supplemented with various concentrations and combinations of BA, Kin, GA3 and NAA.

Well developed shoots (>25 mm long) from proliferating shoot cultures were excised and rooted on MS without auxins or supplemented with different concentrations of IBA.

Explants were cultured for 30-day periods. The cultures were maintained at alternating temperatures of 25/15 oC with a 16-h photoperiod and 35 µmol m-2 s-1 photon flux density. The lower temperature corresponded to the 8-h period of darkness.

Plants obtained from the in vitro rooting phase were washed in water to remove the agar and transferred to 4 cm diameter pots containing sterilised peatmoss. The pots were kept in 35 x 21 cm "mini-propagators" (Sankey Ltd, UK) with a high relative humidity in the same growth chamber for a period of 20 days. The plants and tray surface were sprayed with 2.8 g/l Thiram to retard fungal growth. Subsequently, the plantlets were transferred to a greenhouse.

Twenty replicates were used for each treatment. All experiments on germination, proliferation, rooting and acclimatization were repeated at least twice. Analysis of variance and Duncan's Multiple Range Test were performed on the data. Regression analysis was performed using SAS statistical software (SAS Institute Inc., Cary, NC, USA).

Results and discussion

The presence of hard seed coats is a common feature in species of Cistaceae (Thanos et al. 1992). Therefore, in many species of this family, the scarification of the seed coats is essential to obtain high percentage germination.

Preliminary conventional germination tests carried out in Petri dishes gave the highest germination rates when the seeds were pretreated by immersion in water at 90oC and soaked for 24 h as the water cooled down and incubated at alternating temperatures of 15/25oC (Pérez-García et al., unpublished). In in vitro germination tests, excellent results (83% germination) were obtained when hot water scarification (immersion in water at 90 oC and soaking the seeds for 24 h as the water cooled down) replaced the three water rinses of the standard aseptic procedure.

Table 1. The effect of growth regulators on the number of shoots per explant, the number of nodes per shoot and shoot length after 30 days in culture on MS medium. For each variable, means followed by the same letter are not significantly different (p<0.05).

Growth  regulators  Shoots        Nodes /shoot  Length (mm)   
(mg/l)              /explant                                  

Control             2.0 c         2.4 a         12 a          

0.1 Kin             1.7 c         2.0 ab        10 a          

0.5 Kin             2.0 c         1.8 bc        11 a          

1.0 Kin             1.9 c         1.9 abc       11 a          

0.5 BA              1.7 c         1.3 c         7 b           

1.0 BA              1.7 c         1.7 bc         6 b          

2.0 BA              2.8 bc        1.5 bc        10 a          

5.0 BA              4.0 ab        1.4 bc        10 a          

0.5 BA +  0.1 NAA   3.3 bc        1.4 bc        10 a          

1.0 BA +  0.1 NAA   5.2 a         1.8 bc        10 a          

Responses of shoot tips to various concentrations of growth regulators are summarised in Table 1. The highest number of shoots per explant was attained on MS + 1 mg/l BA + 0.1 mg/l NAA. This value was not significantly greater than that obtained with MS +5 mg/l BA, but it was significantly higher than those obtained with the other media tested. With regard to the number of nodes per shoot and shoot length, highest values were attained on MS without growth regulators. Addition of cytokinins to the media slightly decreased the values for these two parameters. Thus, among the combinations of growth regulators assayed, MS + 1 mg/l BA + 0.1 mg/l NAA was considered optimal for shoot proliferation since it gave a good multiplication rate without interfering with shoot elongation. These results contrast with those of Morte & Honrubia (1992) with Helianthemum almeriense where kinetin was much more effective than BA in inducing shoot proliferation and the best medium for this stage was MS + 0.1 mg/l Kin.

Rooting of shoot explants occurred on MS medium alone or supplemented with varying concentrations of IBA. Within the range of the IBA concentrations tested, the percentage of rooted explants increased with increases in concentration of IBA (Figure 1). The positive linear regression obtained for this variable indicates that a maximum had not been reached. Best results were obtained with MS + 4 mg/l IBA, although higher concentrations of IBA might still provide higher results. Number of roots per explant and root length also increased, in general terms, with concentration of IBA (Table 2). The best response was achieved on MS + 3 mg/l IBA.

Figure 1. The effect of IBA concentration on the percentage of rooted explants in H. polygonoides after 30 days on MS. Line indicates response predicted by regression equation; triangles indicate mean data points. * = significant (p<0.05).

Table 2. The effect of IBA concentration on the number of roots per explant and root length after 30 days in culture on MS. For each variable, means followed by the same letter are not significantly different (p<0.05).

IBA (mg/l)  No.             Root length      
            roots/expl.     (mm)             

0.0         1.2 c           5 b              

1.0         2.6 bc          21 ab            

2.0         2.7 bc          15 ab            

3.0         6.7 a           28 a             

4.0         5.2 ab          26 ab            

An 87% survival rate was obtained in the acclimatisation process when the plantlets were kept in "mini-propagators" and then transferred to the greenhouse.

An effective method for micropropagation of H. polygonoides has been developed. The work on the micropropagation of H. polygonoides was started at a time when the natural habitat of the species was being seriously threatened by the establishment of a water treatment plant at the salt meadow. Fortunately, the location of the water treatment plant has been moved thereby reducing the impact on the natural populations.


This work was supported by CICYT Project AMB 93-0092.


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López-González G. (1993) Helianthemum Mill. In: Castroviejo et al. (Eds.) Flora Ibérica, Vol. III. 365-421. Real Jardín Botánico, CSIC, Madrid.

M'Kada J., Dorion N. & Bigot C. (1991) In vitro propagation of Cistus x purpureus Lam. Sci. Hortic. 46:155-160.

Morte M.A. & Honrubia M. (1992) In vitro propagation of Helianthemum almeriense Pau (Cistaceae). Agronomie 12 :807-809.

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Peinado M., Martínez-Parras J.M., Alcaraz F. & Espuelas I. (1987) Helianthemum polygonoides, a new species of the SE Iberian Peninsula. Candollea 42:361-364.

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