Cultivation of Triphyophyllum peltatum (Dioncophyllaceae), the Part-Time Carnivorous Plant

Received: 5 August, 1998

Keywords: cultivation: tissue culture, Triphyophyllum peltatum -- observations: Triphyophyllum peltatum.

Summary

Cultivation of Triphyophyllum peltatum, a rarely grown part-time carnivorous plant, is presented.

Introduction

Comparatively little is published on Triphyophyllum peltatum (Hutch. & Dalz.) Airy Shaw, a liana widely neglected among carnivorous plant students and growers. Almost nothing has been published on the cultivation of this plant. In the course of our investigations of the secondary metabolites of the plant order Nepenthales, especially of the naphthylisoquinoline alkaloids (Bringmann & Pokorny 1995¹) that are so far known only from two small palaeotropical families, viz. Ancistrocladaceae and Dioncophyllaceae, we became interested in the physiology and biochemistry of these plants. Live material is indispensable for the study of the biosynthesis of the plant natural products. Therefore, efforts were made to obtain living specimens of several species of Ancistrocladus (the only genus of Ancistrocladaceae) and of Triphyophyllum peltatum (one of three monotypic genera of Dioncophyllaceae, and the only carnivorous plant to contain naphthylisoquinolines, Bringmann et al. 1998a). We succeeded in finding seeds and small plants of a number of these species, including T. peltatum. Our experience with germination and cultivation, including in vitro methods, are communicated in this paper.

General

T. peltatum is a high forest liana that grows in coastal rain forests of tropical West Africa (Airy Shaw 1952). Triphyophyllum means "three kinds of leaves," which characterizes this particular genus. During most of its life cycle it is not a carnivorous plant. In its juvenile phase it forms rosettes of undivided lanceolate leaves with obtuse to acute leaf tips. Once the young plant attains a certain age and height (ca. 25-40 cm), on the onset of the next rainy season, carnivorous leaves are formed that have a more or less reduced lamina and a prolonged midrib beset with glandular emergences (Figure 5). These glands secrete a sticky mucilage (Marburger 1979) and capture invertebrates (Green et al. 1979). The uptake of the amino acid l-alanine by the carnivorous leaves of T. peltatum has been demonstrated recently (Bringmann et al. 1998b). Later on, the plant climbs rapidly into the canopy of high trees, attaching to supporting plants by leaves that have two hooks curling towards the leaf base at either side of the leaf tip (Dioncophyllaceae means "plant family with double-hooked leaves"). While the white or pale pink flowers are not spectacular, the seeds are unique within the plant kingdom. Unlike most angiosperm fruits, those of Dioncophyllaceae open prior to seed maturity. These plants have, therefore, been called "secondary gymnosperms" although they are far from being related to the conifers. The large (5-12 cm across), disk-shaped seeds are borne on prolonged, thickened and even woody funiculi (seed stalks) attached to the centre of the disks. The embryo is located close to the point of attachment in a thickened ring of white endosperm approximately 1 cm in diameter (Figure 1), the rest of the seed consists of a thin, papery wing that is slightly striated radially (Figure 2). The detached seeds are able to fly for considerable distances until they reach the floor of the rainforest.

Habitat

Being a West African endemic, T. peltatum grows in the Ivorio-Liberian block of the Western forest massif. It is quite frequently found in the Taï National Park, which belongs to the Guineo-Congolese domain. The dense humid evergreen forest is classified as a "Eremospatha macrocarpa and Diospyros mannii forest," a fundamental type determined by climatic and soil conditions (Guillaumet & Adjanohoun 1971). The component tree species (like Eremospatha macrocarpa (Mann & Wendl.) Wendl., Diospyros mannii Hiern, Diospyros gabunensis Gürke, Maranthes chrysophylla (Oliv.) Prance, Chrysophyllum perpulchrum Mildbr. ex Hutch. & Dalz., Chidlowia sanguinea Hoyle, etc.) are found in other communities, but in lower abundances. The lowland community is a "Diospyros spp. and Mapania spp. forest," as typified by Mapania (Cyperaceae) and Tarrietia utilis (Sprague) Sprague (Sterculiaceae) growing in clay soils.

Above all, the vegetation of the South West of Ivory Coast is marked by the specific endemism of the flora containing particular taxa called "Sassandrians," a term used to describe the species that occur in the shady forests in the West of Ivory Coast between the rivers Sassandra and Cavally. Independent from the presence of T. peltatum, this particular flora contains the following species: Androsiphonia adenostegia Stapf (Passifloraceae), Cassipourea hiotou Aubrév. & Pellegr. (Rhizophoraceae), Cola buntingii Bak. f. (Sterculiaceae), Crinum scillifolium A. Chev. (Amaryllidaceae), Gilbertiodendron robynsianum Aubrév. & Pellegr. (Caesalpiniaceae), Guarea leonensis Hutch. & Dalz. (Meliaceae), Hypolytrum schnellianum Lorougnon (Cyperaceae), Ouratea amplectens (Stapf) Engl. (Ochnaceae), Sciaphila africana A. Chev. (Triuridaceae), Soyauxia grandifolia Gilg & Stapf (Medusandraceae), Thomandersia anachoreta Heine (Acanthaceae), etc. In Sierra Leone and Liberia, T. peltatum exists under the same ecological conditions.

Cultivation of T. peltatum in the Greenhouse

We received uprooted, juvenile but post-carnivorous T. peltatum specimens. In Würzburg, these were planted in fine grain, #2 Lecaton® (from Leca, Pinneberg, Germany) substrate in pots 10 cm wide and 20 cm tall. This substrate does not rot under greenhouse conditions. The high pots provide enough depth for the roots to descend a considerable distance into the substrate. Although they suffered from desiccation when the greenhouse door was lef t open, they grew well for a few years, and produced long shoots with hooked leaves, some of which were several meters long with short shoots that bore normal leaves sprouting from the axils of the hooked leaves. Eventually, however, they all died. Examination of the roots showed that no growth below ground had ever taken place after the plants were uprooted and transported. Similar plants grown in shaded greenhouses in Abidjan showed new root growth after 3 to 6 months, with an average survival rate of 40 to 50%. Especially when the plants are grown in the temperate zone, the sensitive root system should not be disturbed once the plants have been established. This means that there are only two ways to obtain normally growing plants, viz. by seed or by growing rooted plants without removal of the substrate.

Seed is extremely difficult to obtain because it is set during the rainy season, when traveling to the localities of T. peltatum is very troublesome. Furthermore, to harvest seed one must climb high trees since seeds detached by the wind will fly and disappear into the dense ground vegetation. Thus, it took more than ten years before one of us (L.A.A.) finally reached fruiting plants in May 1997. The seeds were sown immediately in Abidjan and in Würzburg under greenhouse conditions. In Abidjan the seeds were planted ca. 3 cm deep in sandy potting soil and watered frequently. The germination rate was ca. 80%. In Würzburg, flat dishes were used with a 2 cm layer of Lecaton®, which was kept permanently wet by a constant water level of 1 cm below the surface of the substrate (maintained by a drainage hole at one side of the dish). The wings of the seeds were removed in order to prevent excessive fungal/algal proliferation, and the seeds were sown on the surface of the substrate. The dishes were kept in a light place without direct sunlight at 22°C and almost 100% humidity. Germination (ca. 50%) took place after a few weeks to several months. The cotyledon tips remained in the seed coat in intimate contact with the endosperm (cryptocotylar germination). The hypocotyl and root pushed their way out of the seed at the former point of attachment of the funiculus. These observations correspond to those by Schmid (1964) with Habropetalum dawei, the only other member of Dioncophyllaceae hitherto reported to have been germinated and grown for several months outside Africa. The growing hypocotyl then lifted the seed up and the cotyledons bent sidewards at their base. The primary leaves sprouted from a narrow slit between the cotyledons where they were attached to the hypocotyl (Figures 3 and 4). These were the first rosette leaves. The rosettes produced leaf after leaf and grew in length without an obvious correlation to the changing (outdoor) seasons of temperate climate. Light intensity was kept constant by artificial lighting (between 8 h and 20 h) so that a mean light sum of 340 klx h was reached daily. Some of the rosette leaves reached a length of more than 30 cm. A one-year old plant with 20 cm long leaves was displayed at the Second Conference of the ICPS in Bonn at the end of May 1998.

In addition, rooted plants of T. peltatum in their substrate were sent to Würzburg in summer 1997. These plants recovered remarkably well from the transport and are still alive. Both the germinated specimens and the plants sent with roots are now being acclimatized to normal warmhouse conditions (average 22°C, occasional minimum of 17°C at about 7 a.m. maximum of 28°C at 6 p.m., average 83% humidity, occasional minimum 53%, maximum 95%, cf. typical plots on Figure 6) because excessive humidity led to algal growth on the leaves and favoured other pests, especially aphids and scale. Larvae of fungus gnats, which are favoured by the required high temperatures and intense watering and which cause severe damage especially on the sensitive roots of young plants, are controlled by continuous treatment with predator nematodes (Nemalogic®, from Sautter & Stepper, Ammerbuch, Germany). The plants are still growing (see Front Cover), and some have attained a size that corresponds to carnivorous specimens in the wild. We have, however, not yet obtained carnivorous leaves. Less than 5% of the seedlings have died. This is the first successful germination of seeds of T. peltatum achieved in cultivation.

in vitro Cultivation of T. peltatum

Axenic in vitro cultivation of plant material facilitates both the propagation of the plants and the investigation of their physiology. There are apparently no reports in the literature on the tissue culture of T. peltatum or any other member of Dioncophyllaceae. We sowed some of our seeds in sterile conditions because greenhouse-grown plant material is difficult to surface-sterilize. The lack of experience with the sterilization of these seeds and the unique occasion of having viable material encouraged us to try several simultaneous approaches. The wings were removed, and the remaining centres were surface-sterilized using 70% ethanol for ten seconds and a sodium hypochlorite solution (13% free chlorine) with one drop of detergent (Triton X-100, from Serva) for 30 minutes followed by three rinses with sterile, distilled water. For some of the seeds we tried to uncover the embryo but the very brittle endosperm and the fragile nature of the embryo complicated this endeavour. The best results with respect to sterility and germination rate were obtained with removal of only the external layers of the seed coat so the white endosperm was not affected. Half of each batch was sown on a different medium. One medium was full-strength Murashige & Skoog medium (1962) supplemented with 3% sucrose and 0.2% Gelrite® and the other one was Seramis® (Effem, Verden/Aller) soaked with MS medium containing 3% sucrose. The media were adjusted to pH 5.8 and filled in 100ml Erlenmeyer flasks. These were autoclaved for 30 minutes at 120ºC and 120kPa.

Germination occurred within three months though the overall percentage was very low (10%). Neither the free embryos nor seeds sown on MS media solidified with Gelrite® germinated. The cryptocotylar germination (see above) was clearly observed in vitro. When the plantlets were about 5cm high their growth slowed. We conclude that the plantlets were not sufficiently supplied with nutrients on the Seramis substrate. The stems were therefore cut above the cotyledons, and the apical parts were aseptically transferred to flasks containing one-fifth strength MS media solidified with 0.2% Gelrite® and containing 3% sucrose. On this medium the plants grew exceptionally well when transplanted monthly to fresh medium (Figure 7). If the plants are not transplanted in this way, they will not grow well.

Currently we are trying to obtain callus cultures, i.e. completely undifferentiated cells using different organs of the specimens as explants. It is hoped that these cultures will grow faster and produce the typical naphthylisoquinoline alkaloids as well as their biosynthetic precursors.

Acknowledgements

The work presented here was supported by the Deutsche Forschungsgemeinschaft (SFB 251 "Ökologie, Physiologie und Biochemie pflanzlicher und tierischer Leistung unter Streß" and Graduiertenkolleg "NMR in vivo und in vitro"). We wish to express our special thanks to Mr. H. Fleischmann, formerly at the Botanical Garden of the University of Würzburg, for having established numerous species of Ancistrocladus and T. peltatum in the greenhouse and for sharing his valuable experience with us.

References

Airy Shaw, H.K. 1952, On the Dioncophyllaceae, a Remarkable New Family of Flowering Plants, Kew Bull., 1952: 327-347.

Bringmann, G., Aké Assi, L., François, G. & Schlauer, J. 1998a, The Alkaloids of Triphyophyllum peltatum (Dioncophyllaceae), Chimia, 52: 18-28.

Bringmann, G., Wenzel, M., Bringmann, H., Schlauer, J., Aké Assi, L. & Haas, F. 1998b, Completing Proof of the Carnivory of Triphyophyllum peltatum (Dioncophyllaceae), Oecologia, submitted.

Bringmann, G., Wenzel, M., Kelly, T. R., Boyd, M. R., Gulakowski, R. J. & Kaminsky, R. (1998c), Octadehydromichellamine, a Structural Analog of the Anti-HIV Michellamines Without Centrochirality, Tetrahedron, submitted.

Bringmann, G. & Pokorny, F. 1995, The Naphthylisoquinoline Alkaloids. In: Cordell, G.A. (ed.): The Alkaloids, vol. 46: 127-271. - New York: Academic Press.

Green, S., Green, T.L. & Heslop-Harrison, Y. 1979, Seasonal Heterophylly and Leaf Gland Features in Triphyophyllum (Dioncophyllaceae), a New Carnivorous Plant Genus, Bot. J. Linn. Soc., 78: 99-116.

Guillaumet, J.L. & Adjanohoun 1971, La végétation de la Côte d’Ivoire, In: Le milieu naturel de la Côte d’Ivoire, Mémoires ORSTOM 50: 157-263; 4 separate maps.

Marburger, J.E. 1979, Glandular Leaf Structure of Triphyophyllum peltatum (Dioncophyllaceae): A "Fly-Paper" Insect Trapper, Am. J. Bot., 66: 404-411.

Murashige, T. & Skoog, F. 1962, A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures, Physiol. Plant., 15: 473-497.

Schmid, R. 1964, Die systematische Stellung der Dioncophyllaceen, Bot. Jahrb. Syst., 83: 1-56.

Footnotes:

¹Part 124 in the series "Acetogenic Isoquinoline Alkaloids". For part 123, see Bringmann et al. 1998c.

Figures:

Front Cover: Juvenile plant of T. peltatum one year after germination, Botanical Garden of Würzburg. (Photo: H. Rischer)