264 S. Afr. J. Bot. 1998, 64(4): 264-266

and van Staden (1992) and Atzmon and van Staden (1994) 5: 133- 138. reported that SWC applied as a soil drench aids in seedling estab­ VAN STADEN, J. & HARTY, A.R. 1988. Cytokinins and adventitious lishment. This is important as root development will affect accli­ root formation. In: Adventitious root fonnation in cuttings, eds. T.D. matization in the greenhouse and seedling establishment in the Davis, B.E. Haissig & N. Scinkilla, pp. 185-204. Dioscordes Press, field. It is suggested that SWC can be successfully used to aid in Portland. acclimatization of in vitro grown plantlets.

Acknowledgements In vitro culture of Eulophia species The University of Natal Research Fund and the Foundation for Research and Development are thanked for financial support. Cultured plants were provided by Mrs L. van Staden. B.G. McAlister and J. van Staden* Natal University Research Un it for Plant Growth and Development. References Department of Botany, University of Natal Pietermaritzburg, Pri­ ALDWORTH. S.J. & VAN STADEN, J. 1987. The effect of seaweed vate Bag X01. Scottsville, 3209 Republic of South Africa. concentrate on seedling transplants. S. Afr. J. Bot. 53: 187- 189. Received 6 February /998; revised 3 June 1998 ATZMON, N. & VAN STADEN, J. 1994. The cll"ect of seaweed con­ centrate on the growth of Pinus pinea seedlings. New Forests 8: 279- Seeds of Eu/ophia cucul!ata, E. streptopetala and E. petersii were 288. placed in aseptic culture and germinated on Murashige and Skoog BECKETT. R.J> . & VAN STADEN, J. 1989. The effect of seaweed con­ medium supplemented with 3% sucrose and 0.01% myo- inositol. centrate on growth and yield of potassium stressed wheal. Plant and E. cucul/ata and E. petersii germinated after 3 months whereas E. Soi/ 116: 20-36. streptopetala took 6 months to germinate. E. streptopetala and E. BLUNDEN, G. & WILDGOOSE, P.B. 1977. The effect of aqueous sea­ petersii had a high percentage of seed germination whereas with weed extract and kinetin on potato yields. J. Sci. Food. Agric. 28: E. cucullata only about 15 seeds germinated from a pod. Multipli­ 121- 125. cation of E. cucu//ata was obtained by cutting the prolocorms in CROUCH, l.J. & VAN STADEN, J. 1991. Evidence for rooting factors half, from which new shoots then developed. These were split fur­ in a seaweed concentrate prepared from Ecklonia maxima. J. Plant.Physiol. 137:319-322. ther so that numerous plants were obtained. Rooting of E. petersii and E. streptopeta!a was increased with the addition of 0.4% acti­ CROUCH. l.J. & VAN STADEN, J. 1992. Effect of seaweed concen­ vated charcoal to the Murashige and Skoog medium. The rooted trate on the establishment and yield of greenhouse tomato plants. J. Appl. !'hyco/. 4:291 - 296. plants were acclimatized successfully in either bark or a sand:soil mixture. CROUCH, l.J., SMITH, M.T.. VAN STADEN, J., LEWIS , M.J. & HOAD, G.V. 1992. Identification of auxins in a commercial seaweed Keywords: Eu/ophia, seeds, in vitro propagation, orchids. concentrate. J. Plant Physiol. 139: 590-594. FINNI E, J.F. & VAN STADEN, J. 1985. The effect of seaweed concen­ • To whom correspondence should be addressed. trate and applied hormones on m vitro cultured tomato roots. ./. Plant Physiol. 120: 215-222. FEATONBY-SMITH, B.C. & VAN STADEN, J. 1983. The ellect of Eu lophia is a very large genus of terrestrial orchids with over 200 seaweed concentrate on the growth of tomato plants in nematode species distributed throughout the tropical and sub-tropical infested soils. Sci. H01·tic. 20: 137-146. regions of the world (Hawkes 1977). In So uth Africa there are 36 FEATONBY-SMITH, D.C. & VAN STADEN. J. 1984a. The effect of species (Schlepe 1966). E. cucullata is a large showy terrestri al seaweed concentrate and fertilizer on growth and the endogenous cytokin in content of Phaseolus vulgaris. S. Aji-. J. Bot. 3: 375- 379. species, one metre or more tall. The pseudobulbs are rounded. FEATONBY-S MITH, B.C. & VAN STADEN, J. 1984b. Identification The leaves are tufted and lanceo late and are 25-30 em long. The and seasonal variation of endogenous cytokinins in Ecklonia maxima inflorescence appears before the leaves. The flowers are pink in (Osbeck) Pupenfus. Bot Mar. 27: 524- 531. colour and are about 3.5 em in length. This plant grows in grass­ HARTMAN. H.T. & KESLER, D.E. 1975. Plant propagation principles lands and on poor pastures, it is widely distributed in tropical and practices. Prentice Hall, Englewood Clift's. Africa (Bechtel eta/. 1981) . JACKSON. M.B.A. & HARNEY, P.W. 1970. Rooting, cofactors, indole E. streptopetala is a robust plant up to 1.5 m tall with well acetic acid und adventiti ous root initiation in mung bean cuttings developed pseudobul bs (Stewart & Hennessy 1981) . The leaves (Phaseolus vulgaris). Can. J. Bot. 48: 943-946. are lanceolate and ribbed, 50 em long and 8 em wide. The inflo­ McALISTER. B.G. & VAN STADEN, J. 1996. In vitro propagation of rescence is many-fl owered. The sepals are green blotched with Kniphojia pauciflora Bak. for conservation purposes. S. Afr. J. Bot. brownish purple inside and the petals are bright yell ow outside 62: 219-221. and pale cream inside with the spur being a purple red. E. strep­ METTING. B., ZIMMERMAN, W.J., CROUCH, I.J & VAN STADEN, topetala grows in woodlands and Eucalyptus plantations and in .1. 1990. Agronomic uses of seaweed and microalgac. In: Introduction long grass at the edge of thickets and amo ngst rocks in montane to applied phycology, ed. I. Akatsuka. SPB Academic publishing, The grassland. It flowers from September to December (Onderstall Hague. 1984). MURASHIGE. T. & SKOOG, F. 1962. A revised medium for rapid E petersii is also a robust plant with large pseudobulbs mostly growth and bio-assays with tobacco tissue cultures. Physiol. P/a/11. 15: 473-497. above ground (La Croix eta!. 1991). The le aves are stiffly succu­ !'ENDERSON. M. 1973. Identifi cation of a cytokinin, lent and are grey green with finely but sharply serrated margins 6-(3-methyl-2-butenylamino) purine in seawater and the effects of (W illi anson 1977). Sepals and petals are green tinged with pur­ cytokinins on brown algae. Plant Physiol. 28: 101- 105. ple-brown markings. The lip is white with purple lamell ae with TA Y, S.A. I3.. MACLEOD, J.K., PALNI, L.M.S. & LETHAM, D.S. the side leaves green with purple veins. E. petersii grows in riv­ 1985. Detection of cytokinins in a seaweed extract. Phytochemist1:v. erine thickets in hot dry areas, usually on a bank well above a 24: 26 11 -2614. river. This plant flowers in November and December. TAY, S.A.B., PALNI, L.M.S. & MACLEOD, J.K. 1987. Identification As Eulophia species are so ld on the traditional medicin e mar­ of cytok inin glucosides in a seaweed extract. J. Plant Growth Regul. kets in South Africa they are becoming rare and steps must be S. Afr. J. Bot. 1998. 64(4) 265

Figure 1 A. Protocorm of E. cucullata producing a shoot. B. E. streptopetafa protocorm masses which can b(! split up to produce shoots and roots. C. E. petersii pscudobulbs with ridged shoots which have developed from the protocorms. D. E. cucultata plantlet in cultur(! with a mass of roots and a few shoots. E. Acclimatized plants of E. petersii. F. Acclimatized plant of£. streptopeta/a beginning to produce a side shoot. taken to conserve them. In vitro culture has been used for this times in sterile water. The explants were cut up and placed onto 4 purpose. Yij eta/. ( 1989) reported on the micropropagation on E. different Murashige and Skoog ( 1962) (MS) media as follows: hormusjii. MS solidified with 1% Unilab agar; MS solidified with 1% Uni- First attempts at tissue culture were made using leaf and pseu­ lab agar and containing 1 mg r1 2,4-0 and 0.5 mg r1 BA; MS dobulb explants of E. petersii and E streptopetala. These liquid without hormones; MS liquid with I mg r 1 2,4-0 and 0.5 explants were removed and sterilized in 3.5% sodium hypochlo­ mg r 1 BA . All media contained 3% sucrose, 0.0 I% myo-inositol rite with a few drops of Tween 20 for I 0 min and then rinsed 3 and were at a pH of 5.8 prior to autoclaving. The liquid cultures 266 S. Afr. J. Bot. 1998, 64(4): 266-268 were placed on a shaker, 60 rpm, in the light. Forty ml medium bark:compost mixture but there was a 60% survival in the was placed in culture flasks with 15 replicates per treatment. sand:soil mixture (Figure IE). E. streptopeta/a acclimatized with Seed pods for the three species were obtained. Some pods had 90% survival in the fine bark (Figure 1F) whi le those in the dehisced and some were still closed. The pods which had not sand:soil and bark:compost mixtures died. E. cucullata had a dehisced were dipped in ethanol and then fl amed. They were 70% survival in the fine bark but died when the other media were then placed in 3.5% sodium hypochlorite for 10 min and after­ used. wards washed in sterile water. The pods were slit and the seeds Although the process for the production of these three species were shaken out so that these were in direct contact with the was slow, more plants could be produced in vitro from a single media. Seeds from the dehisced pods were shaken into a 37 seed pod compared with the number of plants which would be micrometer sieve. These seeds were then sterilized in 1% sodium produced in the natural environment. These cultured plants are hypochlorite for 5 min, and washed 3 times in sterile water. The being maintained successfully and although they have very dif­ seeds were scooped up and dropped into the culture bottles and ferent growth habits in the wild, their growth requirements have dispersed over the surface of the media. Three different media apparently been met. No symbiotic relationship seems to be were used for experiments with seeds: MS with no supplements; needed for the culture of these species. The method described MS supplemented with banana pulp from green bananas (lOOg allows for the production of numerous plants. This will assist in t-1); MS supplemented with 100 ml r 1 deproteinised coconut their conservation. milk. These media were supplemented with 3% sucrose, 0.01% myo- inositol at a pH of 5.8, prior to autoclaving. They were Acknowledgements solidified with 1% agar. The cultures were placed in 16 h light 8 The University of Natal Research Fund and the Foundation for h dark provided by cool white fluorescent tubes at a fluance rate Research Development are thanked for financial support. of 40 ).lmol quanta m-2s-1. Once the protocorm s had developed for all the species they were placed on MS with the addition of 4 References BECHTEL, H., CRIBB, P. & LAUNERT, E. 1981. The manual of culti­ g r I activated charcoal. Three di fferent growing mixes were used in attempts to accli­ vated orchid species. M.T.I. Press, Cambridge. pp. 147- 148. matize the plants of all three species. They were placed in HAWKES, A.D. 1977. Encyclopeadia of cultivated orchids. Faber and sand:soil (2: I v:v); or fine bark: compost (2: 1 v:v) mixtures or in Faber Ltd., London. pp. 208-210. LA CROIX, I.F., LA CROIX, E.A.S. & LA CROIX, T.M. 199 1. straight fin e bark . Half were placed in the mist house with bot­ Orchids of Malawi: the epiphitic and terrestrial orchids from South tom heat (30°C) and half were placed in the greenhouse, watered and East Central Africa. Balkema. Rotterdam . pp. 301- 339. 3 times a week and treated with Kelpak seaweed concentrate ONDERSTALL, J. 1984. Transvaallowveld and escarpment including ( I :500 dilution) every second week. the Kruger National Park. South African wild flower guide. CTP No contamination was obtained with any of the above sterili­ Book Printers, Cape Town. pp. 76. zation methods. E. petersii and E. streptopetala leaves and pseu­ MURASHIGE, T. & SKOOG, f . 1962. A revised medium for rapid dobulbs showed no response on solid or liquid media. The growth and bio-assays with tobacco tissue cultures. Physiologia Pl. explants turned brown and died after 6 weeks. pp.4 73-497. E. petersii and E. cucullata were the first to germinate after 3 SCHLEPE, E.A.C.L.E. 1966. An introduction to South African orchids. months on MS without any supplement. Seeds on the coconut MacDonald, London. pp.28. milk containing medium were slower to germinate and those on STEWART, J. & HENNESSY, E.F. 198 1. Orchids of Africa: A select the banana containing medium did not germinate at all. E. strep­ review. MacMillan South Africa, Johannesburg. pp.l59. topetala germinated after 6 months on MS with no supplements. VIJ, S. P., SOOD, A. & PATHAK, P. 1989. On the utility of rhizome There was very good germination (70-80%) with E. petersii segments in micropropagation Eulophia hormusjii Duth. Orchid Soc. and E. streptopetala with most of the seeds germinating on India. pp. 41-45. . medium with no supplements. E. cucullata seeds, however, had a WILLIANSON, G. 1977. The orchids of South Central Africa. Dent, very low rate of germination ( 1-5%). E. cucullata seeds that did London. pp. 151.:...159. · germinate produced big healthy protocorms (Figure 1A ). Roots and leaves were produced rapidly after the protocorms had developed. E. streptopetala produced numerous protocorm masses (Figure 18) with plantlets being .produced from these In vitro propagation of Drimia robusta protocorms. Although E. petersii was the first to germ inate the Bak. protocorms were slow to develop and slow to produce shoots and roots (Figure I C). This only occurred about 2 months after ger­ mination compared to 2-4 weeks for the other spec ies. G.W. Ngugi1, A.K. JAger* and J . van Staden E. cucu!lata protocorms were cut in half in an attempt to Department of Botany, University of Natal Pietermaritzburg, P/Bag increase the number of explants in culture. Each half sent out X01, Scottsville, 3209 Republic of South Africa new roots and shoots and it was possible to double the numbers 1Present address: Kenya Research Centre for Indigenous Knowl­ in culture at every division of the protocorms. Once the proto­ edge, Centre for Biodiversity, East Africa Herbarium, National corms were divided numerous roots were produced fo llowed by Museums of Kenya, Nairobi, Kenya the production of 2 to 3 shoots (Figure I D). These plants were removed and acclimatized once the root systems were fully Received 2 April 1998; revised 14 June /998 developed. The plantlets of E. petersii and E. streptopetala were placed Drimia robusta Bak. (Hyacinthaceae) is of medicinal importance in on MS media supplemented with 0.4% charcoal in order to South Africa. In vitro bulblet formation was initiated on bulb scale increase rooting. Once the plants had developed small pseudob­ explants or scales attached to the baseplate on Murashige and ulbs they were removed and acclimatized. Plants placed in the Skoog medium supplemented with 0.1 g 1"1 myo-inositol, 30 g r1 mist house died and rotted but those in the green house showed a sucrose, 1 mg 1-1 1-naphthalene acetic acid and 2 mg r1 better survival rate. E. petersii did not survive in the bark and 6-benzylaminopurine. Bulblets were excised from the explant and