In Vitro Propagation of Drosera spp.

Abstract

Additional index words. tissue culture, micropropagation, endangered species, sundew Carnivorous plants are being depleted from their natural environment due to large-scale collection of plants and development of sites that formerly supported carnivorous plant populations (Campbell, 1983; Juniper et al., 1989). Although there are now reputable dealers of carnivorous plants who do not col-lect plants from the wild, some species are already considered threatened or endangered (Ayensu and DeFilipps, 1978). If these plants are to continue to be made available to the public without danger to existing natural stands, it is imperative that alternative meth-ods of production be investigated. In vitro propagation of carnivorous plants is indicated because many plants may be rap-idly produced from a small amount of tissue, thereby minimizing collection from natural populations. Several types of carnivorous plants have been successfully propagated in vitro, including the Venus fly trap Dionaea muscipula Ellis (M. Crehan, personal com-munication) and others (Adams et al., 1979a, 1979b; Carroll, 1982; Mohan Ram et al., 1972). This paper describes a simple method of propagating Drosera spp. in vitro. The sundews, Drosera rotundifolia L, D. capensis L., and D. binata La Billardière, were chosen for this study because of their varying growth characteristics: D. rotundi-folia has a prostrate growth habit with leaves in contact with the soil, and D. binata and D. capensis have upright growth. Plants were obtained from Carnivorous Gardens (Ham-ilton, N.Y.) and grown in 100% sphagnum under fluorescent lights (Philips 40-W Agro-Lite fluorescent bulbs) with a 12-h photo-period. Whole plants and whole leaves were ster-ilized in 10% (v/v) Clorox (0.5% sodium hypochlorite) with one drop of Tween 80 added to each 50 ml of diluted Clorox. Tis-sue was sterilized for 5 min followed by three 5-min sterile distilled water rinses. Whole leaves were used as explants and were placed in 25 × 150-mm culture tubes on 20 ml of medium consisting of Murashige and Skoog mineral salts and vitamins (Murashige and Skoog, 1962) supplemented with 30 g su-crose/liter and solidified with 8 g Difco agar/ liter. Medium was adjusted to pH 5.7 before autoclaving. Because carnivorous plants normally grow in nutrient-poor areas, 1/2, 1/4, and 1/8 normal salt and vitamin con- The cost of pub-lishing this paper was defrayed in part by the pay-ment of page charges. Under postal regulations, this paper therefore must be hereby marked ad-vertisement solely to indicate this fact. 850 centrations (1/2 MS, 1/4 MS, and 1/8 MS, respectively) were examined in addition to full-strength medium. Dilutions of full-strength medium containing vitamins were used for 1/2 MS, 1/4 MS, and 1/8 MS. Ex-plants were grown on media without phy-tohormones and on media containing BA at 0.02 mg·liter -1 and NAA at 0.01 mg·liter -1 , according to Adams et al. (1979b). Cultures were grown at 25C with fluorescent lighting (as described above) under a 16-h photoper-iod. After ≈4 weeks, shoots were visible on leaf explants, apparently forming directly on leaf surfaces without intermediate callus. Leaf explants grown on 1/2 MS with or without hormones produced multiple plantlets that entirely covered the original leaf explant (Fig. 1A). In many cases, too many plantlets were produced to count accurately. All but one leaf explant grown on 1/2 MS and 1/4 MS produced plantlets, but more plantlets were produced on 1/2 MS. Plantlets were pro-duced on 60% and 38% of the explants on 1/8 MS and full-strength medium, respec-tively. Plantlets were readily subcultured on 1/2 MS without hormones. All subcultured D. rotundifolia and D. capensis plantlets pro-duced extensive root systems after 6 to 8 weeks (Fig. 1B), in contrast to the generally weak-rooted plants found in the wild (Juni-per et al., 1989). Only 10% of the D. binata plantlets rooted on medium without hor-mones, whereas 30% rooted on 1/2 MS con-taining BA and NAA. About one-half of the D. rotundifolia and D. capensis cultures pro-duced flowers on 1/2 MS with or without hormones, and 90% of the D. binata cultures produced flowers on 1/2 MS containing hor-mones. D. binata cultures on medium with-out hormones did not produce flowers. Plantlets were removed from culture fol-lowing root development and planted in 100-liter pots in 100% sphagnum or peatmoss. Pots were placed in magenta boxes (Magenta Corp., Chicago) and acclimated by gradually removing the covers, thereby reducing the relative humidity over 4 weeks. Growth con-ditions were the same as for cultures, and mature plants flowered and produced viable seed. About 75% of the original cultures were contaminated. When explants were treated with 20% Clorox for 10 min or 10% Clorox for 20 min, tissue damage was extensive and no regeneration occurred. Occasionally, cul-tures were contaminated in the immediate area surrounding the explant even when the tissue was dead. No clean cultures were ob-tained using whole plants as the tissue source. About 95% of the original D. rotundifolia cultures were contaminated. Drosera rotun-difolia has a prostrate growth habit as com-pared with D. capensis or D. binata, and it is likely that leaves in close proximity to the soil would be infested with more soil-borne organisms than leaves of plants with a more upright growth habit. Contamination prob-lems could also be due to a symbiotic rela-tionship that may exist between some carnivorous plants and certain fungi or bac-teria that may have a role in the digestive process. After a culture appeared to be clean, contamination of subcultures was much re-duced but not eliminated, indicating that mi-croorganisms may remain in intercellular spaces in cultured plants and may occasion-ally be stimulated to resume growth.