Jaroslav Kovác , Department of Genetics and Cell Biology, Palacky University, Slechtitelu 11 77200 Olomouc, Czech Republic
Dianthus arenarius subsp. bohemicus is an endangered endemic from the Czech Republic. Techniques for rapid clonal multiplication and establishment of a genebank of D. arenarius subsp. bohemicus in vitro were developed. Nodal segments excised from plants grown in the field were cultured on modified MS with different growth regulator concentrations. Highest multiplication rate (20:1) was achieved on a medium with 1mg/l BA. On media with IBA or without hormones, the multiplication rate was low (1:7) but rooting percentage was high (up to 83%). Over 65% of shoots rooted in vivo after pulse stimulation with IBA. Rooted plantlets were transferred to soil and, after acclimatization in the glasshouse, to the field. The survival rate was close to 100%, and plants were phenotypically uniform .
BA - benzyladenine, IBA - indole-3-butyric acid, MS - Murashige & Skoog (1962) medium
Tissue cultures can be used for the propagation of endangered and rare species of plants, and therefore can contribute to the protection of plant genetic resources (Fay 1992). The endangered endemic Dianthus arenarius subsp. bohemicus is included in the Red Book of Threatened and Rare Flora of Czechoslovakia. It is found only in the protected reserve "Klenec" in N. Bohemia. Today the population consists of only about 30 clumps. Traditional propagation methods have not been successful. A study was therefore undertaken to develop a
technique for rapid clonal multiplication and to establish an in vitro genebank for this taxon.
Actively growing shoots were excised from plants grown in the field. Single node explants were dipped for 2 minutes in ethanol (70% v/v), soaked for 15 minutes in a 10% dilution of commercial bleach and rinsed three times in sterile distilled water. The explants were trimmed, and single nodes (c. 0.5 cm) were placed in test tubes containing 10ml modified MS + 5mg/l nicotinic acid, 10mg/1 thiamine, 2mg/l glycine, 2.25 mg/l BA and 0.1mg/l IBA, pH 5.5, solidified with 0.7% agar. Cultures were incubated at 25±1ºC under fluorescent, cool white lamps (16h light, 42 µmol/m2/s).
Each axillary bud gave rise to a single extension shoot that was used as the source of nodal explants for shoot multiplication.
After two passages on the initial medium single node explants were inoculated onto 6 media with different concentrations of BA, IBA and macronutrients (Table 1), and the effect of the medium composition on shoot proliferation and rooting was evaluated.
Components Medium H1 H2 H3 H4 H5 H6 Macroelements Full Full Full ½ ½ ½ Salts MS1 strength strength strength strength strength strength Microelements Full Full Full ½ ½ ½ Salts MS1 strength strength strength strength strength strength Nicotinic 5 mg 5 mg 5 mg acid Thiamine - HCL 10 mg 10 mg 10 mg Myo - 100 mg 100 mg 100 mg inositol Glycine 2 mg 2 mg 2 mg 2 mg 2 mg 2 mg Sucrose 30 g 30 g 30 g 15 g 15 g 15 g IBA 0.1 mg 0.1 mg 1.0 mg 1.0 mg BA 2.25 mg 1 mg 1 mg Agar 8 g 8 g 8 g 8 g 8 g 8 g pH 5.5 5.5 5.5 5.5 5.5 5.5
Medium H1 H2 H3 H4 H5 H6 Number of 20.6+/- 20.9+/- 15.1+/- 1.7+/- 1.6+/- 1.7+/- shoots per a 13.7 16.1 10.1 1.0 0.8 0.9 nodal segment The length of 9.8+/- 9.0+/- 12.0+/- 23.1+/- 23.7+/- 26.6+/- shoots (mm) 6.0 4.2 5.7 9.0 9.0 13.4 Number of 2.0+/- 1.9+/- 2.2+/- 3.2+/- 3.7+/- 3.5+/- nodi per a 0.9 0.7 0.7 1.4 1.5 1.3 shoot Percentage of 0 0 0 83.3 68.2 55.3 rooting shoots
Proliferation rate was measured for the second and the third subcultures, each subculture taking 5 weeks on the same medium. Glass jars (150 ml) containing 30 ml of medium and three nodal segments (0.3-0.5 cm long) sealed with transparent plastic caps were used. The cultures were incubated under the same conditions as the initial cultures. For each treatment, 100 explants were used, and the experiment was conducted twice.
A simple method for rooting shoots in non sterile conditions was also tested. After proliferation, single shoots were dipped in 0.05% (w/v) solution of IBA and transferred into perlite or a perlite:sand (1:2) mixture in a closed plastic box.
Resultant plantlets were screened for genetic variability (somaclonal variation) using morphometric studies and esterase isozyme analysis (from polyacrylamide gel electro-phoresis).
For cultures growing on media with 2.25mg/l BA ± 0.1mg/l IBA, the propagation coefficient was high (15.1-20.9). The plants were small, reaching only about 35% of the height of plants cultivated on medium with higher concentrations of IBA. Roots were usually not present, and shoots formed extensive clusters. This effect of relatively high concentration of cytokinins and relatively low concentrations of auxin is common in tissue culture (Pierik 1987) and similar results have been published for other species of Dianthus (Crouch et al., 1993, Kovác, 1992, Kozai et al., 1988).
On media with higher concentrations of IBA or without growth regulators, the propagation coefficient was low (1.6-1.7), and usually each nodal segment produced only one long shoot with 3.2-3.6 nodes.
Rooting percentage on media with higher concentrations of IBA was 55-68% (Table 2). The highest percentage rooting shoots (85%) was obtained on medium without hormones and vitamins and with half the normal concentration of macronutrients and sucrose.
Rooting of shoots originating from media with higher concentration of BA in non sterile conditions was effective. The percentage of rooting plants was 73% for perlite and 65% for a perlite-sand mixture. Rooted plants were easily transferred to soil, and survival rates close to 85% was recorded. Shoots rooted in vitro showed similar survival rate (85%) after their transfer to non-sterile conditions. After two months in the glasshouse, plants were transferred to the field. After two months some plants flowered. A survival rate close to 100% was recorded for the reintroduced plants.
Tissue cultures demonstrated good genetic stability and maintained morphogenetic capacity after one year in cultivation. Preliminary esterase isozyme analyses showed no genetic variability.
Our results show that tissue culture can be used for rapid propagation of D. arenarius subsp. bohemicus. It is now possible to produce a large number of plants, genetically identical to the mother plants, and to reintroduce them to natural habitats. This will aid conservation of this endangered pink.
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Kovác J. (1992) The use of micropropagation in the protection of genofond of Dianthus arenarius subsp. bohemicus. Biol. Plant. 34 (Suppl.):543.
Kozai T., Kubota C. & Watanabe I. (1988) Effects of basal medium composition on the growth of carnation plantlets in auto and mixotrophic tissue culture. Acta Hortic. 230:159-166.Murashige T. & Skoog F. (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant. 15:473-497.
Pierik R.L.M. (1987) Vegetative propagation. Chap. 20 in: In Vitro Culture of Higher Plants. Martinus Nijhoff Publishers. 183-230.