Potential of South African Members of the for New Crop Development

J.G. Niederwieser, M. Terblanche and M.H. Spreeth ARC-Roodeplaat Private Bag X293 Pretoria 0001 . Tel: +27-12 841 9609 Fax: +27-12 8080353. Email: [email protected] and [email protected]

Keywords: , flower crop, genebank

Abstract The results of a scoping study on the potential of the South African Amaryllidaceae for commercialisation are reported. An extensive project to collect as many as possible species of the family in the wild and from other collectors were undertaken. A total of 85 species of 13 genera were obtained, established and maintained in the genebank and observed for a number of years. were evaluated according to appearance of flowers, flowering, vase life of flowers, size of , multiplication, ease of cultivation, etc. Intra- and inter genera crosses were made to determine the potential for breeding of improved varieties. Although many species of this family have spectacular flowers, characteristics such as the size of the flowering bulb, difficulty of cultivation, difficulty to induce flowering in cultivation, limited flower colours within a genus, multiplication rate and vase life present challenging problems with regard to commercialisation. Specific challenges and advantages for some of these genera are discussed briefly. We came to the conclusion that few genera have the potential for further development. Preliminary work on Cyrtanthus indicated that flowering needs to be studied in depth and work at the institute has been suspended until more information is available.

INTRODUCTION The family Amaryllidaceae consists of 17 genera, with approximately 217 species indigenous to Southern Africa (Gibbs Russel, et al., 1985). Species of the family vary from small, for example Strumaria Jacq. Ex Willd., to large for example Brunsvigia Heist. Many species have spectacular flowers that are predominantly red or shades of pink. Some genera have yellow and orange flowers for example Lindl. and Cyrtanthus L.f. . Herb. is a well known genus of the family and has been improved through breeding to one of the most popular flower bulbs. A project to evaluate the potential of the family for new crop development has been initiated in the late 1980’s (Coertze and Louw, 1990). The objectives of the project were to collect a comprehensive collection of species accessions from different climatic areas in the region and to make intra- and intergeneric crosses to develop new cut flowers and pot plants. Coertze and Louw (1990) reported that crosses between species of Cyrtanthus, Nerine and L. resulted in seedlings and that the Nerine hybrids were sterile. Hybrids were obtained from some inter genera crosses through ovule culture. This paper gives an overview of the results of the scoping study which included preliminary characterisation of the genebank accessions, intra and intergeneric crosses and evaluation of the hybrids, especially for the genus Cyrtanthus. Irregular flowering appeared to be the biggest challenge to overcome in this family.

MATERIALS AND METHODS Species accessions were obtained from Botanic Gardens, collectors and some were

Proc. 8th Int. Symp. on Flowerbulbs Eds. G. Littlejohn et al. 359 Acta Hort. 570, ISHS 2002 collected during field trips for which permits were obtained. The identity of plants was confirmed by the National Botanic Institute (Pretoria) where necessary. Plants were maintained in plastic pots in water-cooled green houses at Roodeplaat. Where possible, watering and soil type were similar to those of the natural habitats. The temperature in the green houses varied from approximately 1- 30 oC. Plants were transplanted when the pots became overcrowded as recommended by Du Plessis and Duncan (1989). Cyrtanthus bulbs were transplanted more regularly. Members of the family have four different growing patterns: Summer flowering types with a dormant stage in winter, winter and spring flowering types with a dormant summer, autumn flowering types with a dormant period in summer and evergreen types. This complicated maintenance of the collection as watering and transplanting for different species varied. Pollination was done in the green houses. In general, species that were available in sufficient numbers were used as maternal parent. Pollen of scarce species was collected, dried, stored at 4 oC and used for pollination when maternal parents flowered. Pollen viability was checked regularly through in vitro germination. Cyrtanthus seed were germinated in vitro and seed of other genera were sown as soon as they started to germinate. Pollen tube growth in inter genera crosses was studied by fluorescent microscopy to get a better understanding of the reasons for incompatibility between the genera used. Cyrtanthus hybrid seedlings were grown for four years and evaluation was done in years 2 – 4. Seedlings of inter genus crosses were maintained for a longer period. Hybrids were evaluated for propagation, flowering, appearance of the flowers, stem length and vase life of flowers.

RESULTS AND DISCUSSION

General Species of 13 genera of the Amaryllidaceae have been obtained: (1 species), Ammocharis (1), Boophane Herb. (4), Brunsvigia (6), Clivia Lindl. (3), Crinum L. (11), Cyrtanthus L.f. (38), Gethyllis L. (3), Haemanthus L., (6), Hessea Herb. (1), Nerine Herb. (7), (3) and Strumaria Jacq. Ex Willd. (1). The majority of the genera are used by traditional healers in Southern Africa and have been included in the traditional medicinal nursery at the institute. Ten inter genus crosses resulted in viable seedlings (Table 1). Hybrids of only one cross flowered (C. mooreii x A. belladona) and the flowers were similar to that of C. mooreii. ). Fluorescence microscopy showed that pollen tube growth typically associated with incompatible combinations in most inter genera crosses. In general, the Amaryllidaceae have large bulbs and some of them come into flower only after 6-7 years. The irregular flowering was identified as an important constrain for commercialisation and breeding. Methods for in vitro propagation were published for Cyrtanthus (Niederwieser and Kleynhans, 1997), Amaryllis (De Bruyn, et al., 1992) and Crinum (Slabbert, et al., 1993 & 1995). Attempts to propagate Clivia in vitro were not successful. Gethyllis and Scadoxus were multiplied in vitro using double scale explants. Plants of a number of species were made available to the public through plant sales at the National Botanic Institute (Pretoria) and nurseries. Following an evaluation of the results of the program in 1992, we decided to abandon further inter genera crosses in the Amaryllidaceae and to continue with a scoping study on Cyrtanthus.

Cyrtanthus Genebank A number of 38 species of the approximately 50 described species (Reed and Dyer, 1984) have been obtained and are being maintained in the Roodeplaat genebank. The aim was to obtain accessions from 7 different localities for each species, unless they occur in very restricted areas, for example C. eucallus. A number of endangered species were included in the genebank. C junodii, that was described in 1906 and not seen for 88 years was thought to be extinct. However, it was found during a collection trip to the

360 Drakensberg near Ofcalaco to collect C. thorncroftii. Following identification, the plant was multiplied in vitro and specimens given to the NBI and other collectors. A number of scarce species or ecotypes were propagated through tissue culture, namely C. clavatus, C. staadensis, C. bicolor, C. flanaganii, C. smithiae, C. spirallis, C. tuckii, C. huttonii, and C. junodii. Cyrtanthus species occur naturally in a variety of habitats ranging from the peaks of the Drakensberg (2500- 3000m) (C. erubescence and C. flanaganii), to coastal flats (C. loddigesianus and C. fergusoniae) to the grassy hills of the high veld (C. tuckii and C. contractus) and escarpment (C. junodii and C. thorncroftii). The soil types of the natural habitats vary from sandy to loam and clay. Also, the growing season vary from winter growing, to summer growing and evergreen. We assumed therefor that the conditions in the genebank (especially the temperature, watering and soil type) were not optimal for a number of species and probably had an effect on flowering and propagation of a number of species in the genebank.

Flowering in Cyrtanthus Some species, especially C. brachyscyphus, C. mackenii, C. macowanii and C. staadensis flowered regularly and during specific times of the year. Cyrtanthus elatus is an evergreen species that flowered sporadically throughout the year, but more in the early summer. C. contractus and C. tuckii flower in their natural habitats for a short period in early spring. The plants did not flower regularly under the conditions at Roodeplaat. C. flanaganii and C. erubescence (both growing at high altitudes, 2500 - 3000 m, in the Drakensberg) did not flower at Roodeplaat in seven years. It is possible that the optimal average temperature was too high for these species. Flower initiation and flowering of C. elatus and C. mackenii was described by Slabbert (1997a & 1997b). These species were chosen because they have different growing patterns and plants were available in sufficient numbers. Flower initiation in C. elatus took place throughout the year and up to four inflorescences at different stages of development were observed within a bulb. Bulbs were treated at 10 & 17 oC, but the treatments did not result in increased flowering. In C. mackenii two inflorescences were initiated in the early summer. The primary inflorescence reached anthesis during the following mid winter and the secondary one towards late winter. Flower manipulation of C. mackenii was possible by subjecting the plants to a treatment of 10 C for 3, 6 and 9 weeks after new inflorescences have been initiated in early summer (Slabbert, 1997b). Research needs to be expanded with species such as C. contractus and winter growing species such as C. loddigesianus, as well as promising hybrids. It appears at this stage, that breeding should be aimed at hybrids with seasonal flower initiation as it seems to be difficult to manipulate evergreen plants. Breeding for regular flowering appeared to be possible for example: C flanaganii did not flower at Roodeplaat. However, C. mackenii x C flanaganii and C macowanii x C. flanaganii hybrids did. Furthermore, preliminary observation indicated that some species could be associated with regular flowering when used in crosses, for example: C. brachyscyphus, C. galpinii, C. eucallus and, C. macowanii. Other species appeared to be associated with poor flowering, for example: C. montanus and C. elatus.

Propagation in Cyrtanthus The propagation rate of Cyrtanthus species through daughter bulb (off set) formation varied considerably. C. montanus formed many dormant off sets throughout the growing season. C. mackenii, C. elatus, and C. eucallus, formed enough off sets per year to result in large numbers of plants in the genebank after three years. C. herrei, C. obliquus and C. falcatus formed off sets occasionally and C. staadensis appeared to propagate primarily through seed. Preliminary results appeared to indicate that C. elatus and C. montanus were good parents for off set production, whereas C. galpinii, C. contractus and C . sanguineus were not. Tissue culture proved to be a viable method of propagation for Cyrtanthus

361 (Niederwieser and Kleynhans, 1998). All 18 species tested, propagated through the double scale method. Approximately 50% of these species formed adventitious buds on single scale explants. Scarce species such as C. carneus, C. erubescence, C. flanagani, C. guthrieae, C. junodii, C. leucanthus, C. thorncroftii and C. ventricosus, as well as hybrid selections were propagated in vitro. Double scaling in vivo appeared to be a useful method to propagate selections (Slabbert, unpublished). C. elatus, C. mackenii and C. brachyscyphus produced 30, 18 and 14 bulblets per bulb respectively within 12 weeks, using double scale explants that were placed in a composted bark medium at 20C.

Cyrtanthus Hybrids Ten species were used as maternal parents and 25 as pollen parents (Table 2). A total number of 320 inter species crosses were made and more than 7000 F1 hybrid seedlings were obtained from 106 (46%) of the crosses (Table 3). Of the species that were used most often as maternal parent (Table 3), C. brachyscyphus, C. elatus, C. eucallus, C. macowanii and C. eucallus gave useful results and C. breviflorus, C. galpinii, and C. sanguineus gave poor results. Of the species most often used as pollen parent, C. carneus, C. erubescence, C. fergusoniae and C. thorncroftii appeared to give poor results and C. flanaganii, C. galpinii, C. loddigesianus, C. montanus, C. obliquus, and C. sanguineus appeared to give useful results. The major problem encountered with the hybrids was that the majority did not flower. For example: Hybrid seedlings were obtained from 94 inter species crosses in 1993. Of these, 62 never flowered in four years. Of nine crosses, more than 25% of the seedlings flowered and of the remaining 23 crosses, less than 25% of the seedlings flowered within the four years of observation. Propagation appeared to be less of a problem. More than 50% of the crosses resulted in hybrids with a satisfactory to good propagation rate. However, 40% of the 1993 crosses resulted in hybrids with poor propagation (one or less off sets per year). Other poor characteristics of Cyrtanthus hybrids were identified namely: i. Tubular shaped, hanging flowers (similar to that of C. mackenii and C. brachyscyphus), ii. Short inflorescences (less than 30 cm), iii. Many hybrids did not have firm stems and the stems of other hybrids broke easily. Good characteristics of hybrids included: i. Relatively small bulbs, ii. More than 50% of the crosses resulted in hybrids with an acceptable rate of propagation iii. Flower colour of most hybrids were bright and mostly red. iv. Hybrids of C. flanaganii crosses, had a pleasant fragrance and v. The vase life of flowers were comparable to that of Nerine.

CONCLUSION An extensive genebank of the Amaryllidaceae has been established. Inter genera crosses within the family resulted in relatively few hybrids of which only one flowered. As a result of the long breeding cycle required for most species, further breeding on most of the genera in the family were not feasible for Roodeplaat. Methods for in vitro propagation of various genera were developed and applied to increase scarce plants in the genebank and to make popular plants available to the public. The scoping study on the genus Cyrtanthus, resulted in a large number of seedlings (7000+) from more than 300 inter species crosses. The biggest constrain for continued breeding and hybrid evaluation is thought to be irregular flowering. Inflorescences were continuously initiated in the evergreen C. elatus and manipulation of flowering was not possible. In C. mackenii, floral initiation took place in early summer and flower manipulation appeared possible. Further breeding on Cyrtanthus at Roodeplaat had been suspended until more information is available on flowering.

ACKNOWLEDGEMENTS D. Mabele and S. Mziza for technical assistance and maintenance of the genebank. A Coertze for collection of various species. R. Slabbert for development of the double scaling method and preliminary trials on flower manipulation. National Botanic Institute

362 (Pretoria) for identification of new accessions to the genebank. Various persons who donated plants or offered assistance during field trips.

Literature Cited Coertze, A.F. and Louw, E. 1990. The breeding of interspecies and intergenera hybrids in the Amaryllidaceae. Acta Hort. 266, 349-352 De Bruyn, M. H., Ferreira, D.I., Slabbert, M.M. and Pretorius, J. 1992. In vitro propagation of Amaryllis belladona. Pl. Cell Tis. Org. Cult. 31, 179-184. Du Plessis, N. and Duncan, G. 1989. Bulbous plants of Southern Africa. Tafelberg Publishers, Cape Town. South Africa. 192 pp. Gibbs Russel, G.E.G., Read, C., van Rooy, J. and Smook, L. 1985. List of species of Southern African plants. Ed.2 Part 1. Mem. Bot. Survey of S. Afr. 51, 109 -113. Niederwieser, J.G. and Kleynhans, R. 1998. Adventitious bud formation on bulb scales of Cyrtanthus species (Amaryllidaceae). J. Sth. Afr. Soc. Hort. Sci 8, 24-26. Reid, C. and Dyer, R.A. 1984. A review of the Southern African species of Cyrtanthus. The American Plant :Life Society, California. Pp 104 Slabbert, M.M, De Bruyn, M.H., Ferreira, D.I. and Pretorius, J. 1993. Regeneration of bulblets from twin scales of Crinum macowanii in vitro. Pl. Cell Tis. Org. Cult. 33, 133-141. Slabbert, M.M., De Bruyn, M.H., Ferreira, D.I. and Pretorius, J. 1995. Adventitious in vitro plantlet formation from immature floral stems of Crinum macowanii. Pl.Cell Tis. Org. Cult. 43, 51-57. Slabbert, M.M., 1997a. Inflorescence initiation and development in Cyrtanthus elatus (Jacq. Traub). Sci. Hort. 69, 61-71. Slabbert, M.M. 1997b. Flower bud initiation and development in Cyrtanthus mackenii (Hook F.) as influenced by temperature. J. Sth. Afr. Soc. Hort. Sci. 58-61.

Tables

Table 1. List of inter genera crosses in the Amaryllidaceae that resulted in viable hybrid seedlings.

Number of Maternal parent Pollen parent seedlings Notes Amaryllis belladona Crinum acaule 3 Did not flower A. belladona C. mooreii 1 Did not flower Brunsvigia orientalis A. belladona 109 Did not flower Cyrtanthus mooreii A. belladona 11 Produced flowers similar to C. mooreii Nerine augustifolia A. belladona 8 Did not flower N. bowdenii A. belladona 5 Did not flower N. bowdenii A. belladona 7 Did not flower N. bowdenii B. orientalis 5 Did not flower N. laticoma C. montanus 1 Did not flower N. sarniensis B. orientalis 2 Did not flower

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Table 2. Cyrtanthus species used most often for inter species crosses, with notes on characteristics that are relevant to the development of a new crop

SPECIES FAVOURABLE POOR CHARACTERISTICS CHARACTERISTICS C. attenuatus Yellow flowers Hanging, tubular flowers C. brachyscyphus Small bulbs, flowers regularly Small, hanging flowers, tubular shape C. breviflorus Yellow, open flowers Flowers irregularly, small plant C carneus Tall flower stem, pink flowers Poor propagation C. contractus Tall stem, large flowers, bright Flowers irregularly, semi-tubular colour flower, poor propagation C. elatus Spectacular flowers, open, Flowers irregularly facing up, propagate well. C. erubescence Tall stem, pink flowers Flowers very irregularly, difficult to grow, poor propagation C. eucallus Open flowers, propagate well Small plant, few flowers/stem C. falcatus Red flowers Flowers irregularly, poor propagation C. flanaganii Yellow flower, fragrant, Flowers very irregularly, semi- tubular flower, poor propagation C. fergusoniae Tall stem, red flowers Difficult to grow C. galpinii Spectacular flowers Small plant, poor propagation, single flower/stem C. guthrieae Bright red flowers Difficult to grow, few flowers C. herrei Tall, strong stem Flowers irregularly C. loddigesianus Open flower, facing up, white Small plant, few flowers, poor propagation C. macowanii Small bulbs, flower regularly, Tubular, hanging flowers propagate well C. mackenii Small bulbs, flower regularly, Tubular, hanging flowers various colours, propagate well C. montanus Open flowers, facing up, good Flowers irregularly, produce propagation abundance of off sets C. obliquus Tall, strong stem, large multi Flowers irregularly, poor colour flowers, propagation C sanguineus Spectacular, large flowers Poor propagation C. smithiae Attractive flower Flowers irregularly, single flower/stem C. spiralis Attractive flower Poor propagation, flowers irregularly C. staadensis Unique flower shape Poor propagation, small plant C thorncroftii Pink, open flower Small plant C. tuckii Tall, firm stem Flowers irregularly

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Table 3. Inter species crosses in Cyrtanthus

ae

C. attenuatus C. brachyscyphus C. breviflorus C. carneus C. contractus C. elatus C. erubescence C. eucallus C. falcatus C. flanaganii C. fergusoni C. galpinii C. guthrieae C. herrei C. huttonii C. loddigesianus C. macowanii C. mackenii C. montanus C. obliquus C. sanguineus C. spiralis C. staadensis C. thorncroftii C. tuckii C brachyscyphus b n r r b b b r b b r b r b r b b b b r r b C. breviflorus r n r r r r r C. elatus b b b n b b b r b r b r b b b b b r b r b C. eucallus b r b r b b r b b b b r b b b b r b r C. galpinii r r r b r r r n r r r r b r b r r r C. loddigesianus b r r r r r r C. macowanii b b b b b b r a r b r b b b b b r a C. mackenii b b b r b b r r b b r a r r r a n a a a r r r C. montanus b b b b r b r a a a a a a a a a a a n b b b b C. sanguineus r r b b r r r b r r a r r r n r

365