Valerie C. Pence ¹ & Victor G. Soukup ²
¹ Center for Reproduction of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA
² Herbarium, Department of Biological Sciences, Univ. of Cincinnati, Cincinnati, OH 45221, USA
¹ Author to whom correspondence should be addressed
Bud tissue of Trillium persistens was cultured on half-strength Murashige & Skoog (1962) medium (½MS) + 3% sucrose, 1 mg/l BAP and 1 mg/l NAA, on which they formed nodular, shoot-producing mini-rhizomes (MRs). These could be subcultured and maintained on a similar medium lacking growth regulators. Separated shoots rooted on the same medium producing plants which were transferred to soil.
Trillium persistens Duncan (Trilliaceae), or persistent trillium, is a rare native woodland species which has been listed as nationally endangered (U.S. Dept. of the Interior, Fish and Wildlife Service, 1978) (Figure 1). First described in 1971 (Duncan, et al., 1971), it is known from only 4 populations in Georgia and South Carolina. The species is threatened by habitat loss to development and logging and is also of interest to wildflower collectors (U.S. Fish and Wildlife Service, 1984). Techniques which could provide vegetatively propagated material for commercial use and for possible reintroduction are, therefore, of interest. The goal of these studies was to apply techniques which had been developed for the in vitro propagation of Trillium spp. (Pence & Soukup, 1986; 1993) to the rare species, T. persistens.
Two dormant rhizomes of T. persistens were collected by Mr Thomas S. Patrick from the South Carolina-Georgia border site, with authorization from the South Carolina Wildlife and Marine Resources Department Heritage Trust Program.
Buds were dissected from the rhizome and surface sterilized in a 10% (v/v) dilution of commercial sodium hypochlorite for 10 minutes, followed by two rinses of sterile distilled water. Later resterilization was done using a 5% (v/v) dilution of sodium hypochlorite. The buds were then cut into 4-5 small pieces and cultured on ½MS + 1 mg/l naphthaleneactic acid (NAA), 1 mg/l benzyl aminopurine (BAP), 3% sucrose, and 0.8% agar in 60 mm x 15 mm sterile disposable culture dishes, approximately 15 ml/dish. Cultures were maintained in a growth room at 26°C with 16h light, under fluorescent cool white lights.
After 12 days, all pieces were transferred to fresh medium. Because of a light bacterial contamination, all pieces were resterilized at 19 days and again transferred to fresh medium. Once MRs were established, the tissues were subcultured onto ½MS medium without growth regulators. In vitro produced shoots were excised and placed onto the same medium in 25 mm x 150 mm culture tubes, 25 ml/tube, for root initiation. Rooted shoots were transplanted to potting soil. The plants were placed in a greenhouse either under a misting system, or under plastic bags. When bags were used, holes were punched in the bags to gradually acclimate the plants.
With rare species there is often not a sufficient amount of tissue available for experimentation. In this case, a growth regulator combination which was successful with a related species, T. erectum, was selected for use. In addition, half-strength salts were used, which had been found to be beneficial to T. grandiflorum, and not detrimental to other Trillium spp. which have been tested (Pence & Soukup, 1993).
With this medium, two of the nine resterilized bud pieces gave rise to swollen, smooth-surfaced, cream-coloured tissues resembling rhizome tissue, designated mini-rhizome (MR) tissues. Each MR gave rise to a single shoot and, in addition, produced new MRs which gave rise to new shoots. Once the MR tissue was initiated from the original explant on the hormone-containing medium, further growth was autonomous and continued in the absence of growth regulators. Shoots were rooted in tubes on medium lacking growth regulators, and once roots had formed these were transferred to pots.
The production of MRs by T. persistens in vitro is similar to the response observed with bud, leaf, and stem explants from other Trillium spp. in the presence of auxin and cytokinin (Pence & Soukup, 1986; 1993). The direct formation of MR tissue is also similar to the direct formation of bulblets in other liliaceous species (Robb, 1957; Pierek & Woets, 1971).
Compared with other tissue culture systems, Trillium grows slowly in vitro. It may take 4-8 months for a piece of MR tissue to produce new MRs and for those to develop shoots which can be isolated for rooting. Rooting may then take another 6-8 months. However, young Trillium plants can be produced in tissue culture in 2-3 years less than from seed. In addition, once MR cultures are initiated, many plants may be propagated from a single source. During the course of these studies, more than 230 plants were obtained from two original source plants. This is the approximate equivalent of one-sixth of all the plants remaining in the wild.
The authors gratefully acknowledge Dr John L. Caruso, Department of Biological Sciences, University of Cincinnati, in whose laboratory much of this work was done.
Duncan W.H., Garst J.F. & Neece, G.A. (1971) Trillium persistens (Liliaceae), a new pedicellate flowered species from northeastern Georgia and adjacent North [sic] Carolina. Rhodora 73:244 -248.
Murashige T. & Skoog F. (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15:473- 497.
Pence V.C. & Soukup V.G. (1986) Plant regeneration from Trillium spp. in vitro. HortSci. 21:1211-1213
Pence V.C. & Soukup V.G. (1993) Factors affecting the initiation of mini-rhizomes from T. erectum and T. grandiflorum tissues in vitro. Plant Cell Tiss. Org. Cult. 35:229-235.
Pierik, R.L.M. & Woets, J. (1971) Regeneration of isolated bulb scale segments of hyacinth. Acta Hortic 23:423-427.
Robb, S.M. (1957) The culture of excised tissue from bulb scales of Lilium speciosum Thun. J. Exper. Bot 8:348-352.
U.S. Fish & Wildlife Service (1984) Persistant Trillium Recovery Plan. U.S. Fish and Wildlife Service, Atlanta, Georgia. 69 pp.
U.S. Fish & Wildlife Service (1978) Determination that 11 plant taxa are endangered species and 2 plant taxa are threatened species. Fed. Reg. 43:17910-17916.