Biological Research Center, 48 Republicii Street, 3400 Cluj - Napoca, Romania.
Methods for in vitro propagation of Romanian populations of Leontopodium alpinum were developed. Although the number of shoots produced was lower than some other media, MS + corn extract (medium 4) was considered to give the best microplants.
Several plant species have disappeared from the Romanian flora and many others are on the verge of extinction. This is a cause of increasing concern for conservation of rare and endangered species important from a scientific, economic or landscape point of view.
Methods of conserving and restoring natural resources have been given much attention lately. Among these, in vitro micropropagation for obtaining a large number of plants in order to repopulate areas has aroused great interest.
Prof. Alexandru Borza (1887-1971), the founder of the Botanic Garden of the University of ClujNapoca, established a list of plants suggested as 'natural monuments' as early as 1924. These included Leontopodium alpinum (L.) Cass. (Compositae) (Ionel et al., 1986). This species occurs in the Pyrenees, the Alps, the Carpathians, the Balkans and Central and E. Asia. Although an alpine plant, in Romania it can be found in valleys eg. in the lower Râmet Gorges, probably the lowest site for this species in Europe (about 490 m). Being extremely ornamental it has been excessively collected in Romania and has become rare, a symbol of nature preservation, a plant with a glorious past and an endangered future.
Hook (1993) studied L. alpinum looking at micropropagation, secondary metabolite production and medicinal value, stating that micropropagation could be the best source of biological material needed for secondary metabolite extraction. The present study deals with conservation by in vitro propagation of L. alpinum.
Samples for use as explants were collected in the Râmet Gorges. The explants for in vitro culture were extremely young inflorescences from which isolated flowers (about 12 mm) were detached. The inflorescenses were sterilized in 5% calcium hypochlorite for 30 minutes and rinsed repeatedly with sterile water. The explants were then incubated on a range of sterile media (Ll - L6) based on MS (Murashige & Skoog, 1962), with half or full strength mineral components and growth regulators. These were supplemented with natural extracts or high concentrations of substances such as ammonium nitrate, sodium monophosphate, adenine sulphate etc. The composition of the media is given in table 1.
Table 1. Media used for the micropropagation of L. alpinum.
Components Units Ll L2 L3 L4 L5 L6
Macroelements ½MS ½MS MS MS MS MS Microelements ½MS ½MS MS MS MS MS FeEDTA ½MS ½MS MS MS MS MS Myo-inositol mg/1 50 50 100 100 100 100 Thiamine HC1 mg/1 0.1 0.1 1 1 1 1 Pyridoxine mg/1 0.1 0.1 1 1 1 1 HCl Nicotinic mg/1 0.1 0.1 1 1 1 1 acid Sucrose g/l 25 25 30 30 30 30 Agar g/l 6 6 6 6 6 6 IAA mg/1 - - 2 - - - IBA mg/1 - - - - 0.1 0.5 BA mg/1 - - 2 - 0.1 0.1
charcoal (g/1) - 3 - - - - adenine sulphate (mg/l) - - 40 - - - corn extract (mg/l) - - - 1 - - NH4NO3 mg/1) - - - - 825 - sodium (mg/l) - - - - - 170 monophosphate pH 5.8 5.8 5.7 6.1 6.1 5.7
MS = Murashige & Skoog Medium; MS = half MS components
The cultures were maintained in an 8 h photoperiod (2000 lux) at 24°C. Cultures were initiated at the beginning of July and the rate of neoplant formation (number and height) and the development of the root system (number, length and vigour) were recorded after 8 weeks.
Average values of the parameters measured are given in Table 2.
Table 2. Effects of medium composition on in vitro propagation of L. alpinum.
Medium Neoplants Root development
No. Height No. Length (cm) (cm) L1 5 0.8 4 0.6 L2 32 3.8 8 1.5 L3 84 0.7 - - L4 8 1.8 8 5.0 L5 23 0.3 2 1.0 L6 48 1.0 1 0.3
On L1 , few new shoots were formed (about 5 neoplants/explant, c. 1 cm high) and these had a fragile root system (3 roots of c. 0.5 cm). It is noteworthy that neoplants can be formed and root development can occur on a medium lacking growth regulators and having half the normal mineral components. We regard this as advantageous for in vitro propagation, as the cost of such a medium is low.
Charcoal in L2 promoted propagation: over 30 neoplants c. 3.5 cm high were obtained per explant. On this medium development of neoplants and roots was uniform for all explants. The neoplants each had 5-8 roots c. 1 cm long. Completely formed and well elongated neoplants were an excellent source of minishoots, ensuring an increased rate of propagation.
The highest rate of propagation was recorded on L3 with over 85 neoplants/explant. These were short (0.50.7 cm) and lacked roots. The number of neoplants obtained on this medium represents a significant source of material for propagation. When subcultured onto medium containing charcoal (L2) they developed roots and multiplied. After c. 3 months the initial minishoot was surrounded by 2030 new plantlets. These were small initially, but developed normally later.
The corn extract in L4 enhanced propagation slightly, giving 8 neoplants 2 cm high, which were well rooted (8 roots, 5 cm long) and bright green in colour. Although the percentage was low, the complete development of plantlets with regard to their height, number and length of roots accounts for our using this medium for other species as well.
L5 also enhanced propagation (over 20 neoplants/explant). The neoplants were short and poorly rooted and their leaves were light green or almost chlorotic, possibly due to the concentration of ammonium nitrate in this medium.
The presence of sodium monophosphate together with IBA and BA in L6 was favorable to propagation. Up to 40 neoplants/explant were obtained, but root development was poor (roots 0.2-0.3 cm long occurred sporadically). It also promoted callus formation on the lower part of neoplants, which hindered root formation. The plantlets were detached from their original clumps and transferred to L2 on which they developed roots in less than 3 weeks.
The acclimatization percentage for ex vitro plantlets grown on media L1, L2 and L3 amounted to 85%. Although the number of resulting neoplants was relatively low, the acclimatization percentage was high due to the good root system. Unrooted neoplants from L3 or poorly rooted ones from L5 and L6 were subcultured in vitro onto L2. In about 3 weeks they elongated and developed roots and were then weaned with a high percentage success.
In vitro regeneration of L. alpinum can occur on media lacking growth regulators and with half the mineral components without (Ll) or with 3 g/1 charcoal (L2). Thus in vitro culture and propagation are possible at low cost.
The presence of adenine sulphate in the medium enhanced propagation of L. alpinum with up to 80 neoplants/explants being produced, but inhibited the development of the root system even when an auxin was added (L3).
The addition of 1 mg/l corn extract gave well organised regenerated neoplantlets, with the best root system and the highest acclimatization percentage.
Sodium monophosphate and ammonium nitrate in the presence of IBA and BA also enhanced the production of explants obtained from L. alpinum inflorescences (20-48 neoplantlets/explant), but inhibited development of an adequate root system.
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