Problems and Breeding Strategies for the Development of New

R. Kleynhans F.L. Hancke ARC-Roodeplaat Present Address: Private Bag X293 Boekenhoutskloof Pretoria, 0001 P.O. Box 902 South Africa Cullinan, 1000 Tel:+27-12-8419611 South Africa Fax:+27-12-8080353 Tel:+27-12-7320076 e-mail: [email protected] Fax:+27-12-7320076 e-mail: [email protected]

Keywords: breeding, flower , Lachenalia

Abstract Lachenalia is a bulbous geophyte endemic to South Africa and Namibia. Initial breeding on this genus was started in 1965 with the aim of developing new pot cultivars. The first cultivars resulting from the breeding program have been commercialized in Holland over the last three years. New information on market demands, crop related problems and undesirable characteristics of the current cultivars are coming forth each year. Marketing new cultivars on a regular basis keeps the consumer interested in the product. Careful planning is needed to satisfy this demand as it takes a minimum of 12 years from making a cross until the is available in the market. Compiling good breeding strategies and contingency plans is thus of the utmost importance. This includes, getting basic information through research, incorporating new crop production information into the selection procedures, regular contact with all commercial growers involved, utilizing different breeding techniques and a intuition of what the market wants in 15 years time.

INTRODUCTION The ARC-Roodeplaat realised the potential of the South African bulbous and started to develop some endemic as commercial pot plants and cut flowers several years ago. One of the genera selected for pot plant breeding was Lachenalia (Jacq.f. ex Murray). Hundreds of crosses have been made and several reports have been published on the breeding work done by the ARC-Roodeplaat (Lubbinge, 1980; Malan et al., 1983; Lubbinge et al., 1983 a, b, c, d; Ferreira and Hancke, 1985; Hancke and Coertze, 1988; Coertze et al., 1992). However it was only during the late 90’s that the first commercial pot plants were sold in the Netherlands, offering the Dutch growers something completely new, interesting and different. To hold the interest of the market in Lachenalia it is essential that new cultivars be made available continuously. Satisfying in this condition, however, proves to be a time-consuming and demanding task. The genus has a three-year breeding cycle from seed to flowering. It takes a minimum of 8-9 years from making a cross until a successful cultivar can be released. This period includes all evaluation and multiplication steps in the hybrid evaluation process. Following release, there is a further four-year period before the cultivar is available in the market. The supply of disease free mother material and commercial multiplication of the crop spans this period. A minimum of 12-13 years is thus needed for the development of a new cultivar. The current objective is two new cultivars every three years. These cultivars must conform to market requirements. Knowledge of market requirements and preferences is important for planning strategies to satisfy market demand. Regular contact and contributions from committed partners in the different export countries (Europe and USA) assist the breeder in planning breeding strategies to incorporate the latest trends in the markets. New partnerships are needed to test the potential of the product in other market sectors. The time constraints for the release of new cultivars and the changing and differing Proc.market 8th Int.requirements Symp. on Flowerbulbs influences the breeding strategies. These factors must be taken into Eds. G. Littlejohn et al. Acta Hort. 570, ISHS 2002 233 account when other problems are addressed. Problems associated with the supply and demand for new Lachenalia cultivars will be discussed in this paper.

GENEBANK VARIATION Lachenalia has a wonderful variety of expression of characteristics to be utilized in breeding. Firstly the multitude of colors and color combinations and secondly the three basic flower forms; namely the hyacinth, the bell shape and the tubular shape flowers. The inflorescence varies from bright and robust to small and dainty. Other interesting characteristics are spots and striped markings on the leaf, which can add to the aesthetic appearance of the end product. A breeder’s dream in terms of possibilities but also a nightmare, if specific and focused objectives are not set. Firstly the species delimitation within the genus is often poor and the absence of a taxonomic key for all the species leads to frequent misidentification of specimens (Crosby, 1986). Proper taxonomic information and information on the relationship between and within species is of utmost importance for proper planning in the breeding program. Research on these aspects is currently underway (Spies et al., 2000, Van Rooyen et al., 2000). Secondly the variation in the genebank is of little use if the accessions have not been characterized properly. An evaluation system has been developed specifically for the breeding of Lachenalia pot plants. Each accession entering the genebank is characterized for certain breeding characteristics such as flower form, flower color, inflorescence/peduncle ratio, firmness of the peduncle, number of flowers and natural multiplication potential. Twenty-five phenotypic characteristics are noted and other information such as chromosome numbers; pollen fertility and self-incompatibility are added, as they become available. This computerized database is utilized when crossing strategies are planned. Thirdly the differing flower sizes causes problems in marketing procedures. By utilizing the variation in the genebank, small and large flowered hybrids have been developed (Lubbinge, 1980 and unpublished results). The cultivars currently available in the market all have relative large flowers and inflorescences. The different colors and flower forms aesthetically compliment each other. When cultivars are sold, they are thus presented in mixed trays. The smaller flowered hybrids, however, do not compliment the existing hybrids. A range of small flowered cultivars is thus required for the same type of marketing. Fourthly the variation within species must be utilized. Lubbinge (1980) mentioned that the varieties of species from different localities do not necessarily give the same results when crosses are made. The large variation within species in terms of chromosome numbers and appearances necessitate the collection of more than one accession of each species. Combining different accessions of the same two species can give different results in terms of obtaining seed set (Lubbinge, 1980; and Table 1) and also in terms of the appearance of hybrids. The same is true for reciprocal combinations (Lubbinge 1980 and Table 1). In the breeding strategy it is thus important to combine different accessions to utilise the variation within species. Making intra-species crosses is another strategy to utilise the variation within species. The resulting intra-species hybrids are then used in further interspecies crosses instead of the pure species accessions. Creating these intra-species genepools has the added advantage of braking down incompatibility barriers (see incompatibility). Effective genepools can however, only be developed if the amount of variation within species is known. Appearances, cytogenetics and molecular systematics can be used to determine the intra-species variation. L. bulbifera (Cyrillo) Engl. for example forms a polyploid complex. Accessions with the same chromosome number, however, are not necessarily closely related (Kleynhans and Spies 2000).

BASIC RESEARCH INFORMATION Obtaining basic research information on a number of aspects is becoming more and more important to the Lachenalia breeding program. The conventional crosses that are “easy” have been made. Developing new hybrids in the future will require more applied methods. Currently the main limitation in obtaining the required information is funding. Strategies focussing on research contracts with universities address this problem to some extent. 234

Incompatibility and Crossability The variation in the genus also causes a number of isolation barriers (Lubbinge, 1980). Variation in the flowering time of species (April to November) in South Africa, makes certain crossing combinations difficult. Pollen can however, be stored successfully to overcome this isolation barrier (Lubbinge, 1980). Other isolation barriers include incompatibility. Self-incompatibility exists within most Lachenalia species accessions (Table 2). Most species accessions do not produce seed when they are self-pollinated (Table 2). Intra-species crosses between different accessions usually overcome this problem (Table 1), but even then it is important to combine accessions collected from different areas. Poor crossability between species occurs (Table 1), resulting in failure of crosses and non-viable seed. Of all the failed F1 interspecies crosses made at Roodeplaat, 50% did not succeed because of a lack of seed set, 44% because of non-viable seeds and 6% because of seedling mortality. Basic research on incompatibility is needed to understand and overcome these problems. Initial research showed that the incompatibility could be attributed to different causes: - Flower length and the inability of pollen of shorter flowers to grow down the style of longer flowers (Lubbinge, 1980). The reciprocal cross overcomes this problem in some cases, but does not guarantee success (Table 1). The cut-style method has been used successfully to produce hybrids between L. bulbifera (large flower) and L. mutabilis Sweet (small flower). This method must be tested on other interspecies combinations and optimized if necessary. - Failure of the pollen tube to reach the ovary. Initial work on pollen tube growth showed that very few pollen tubes of certain L. aloides (L.f.) Engl. accessions reach the ovary. Pollen tube growth stopped either below the stigma or at the bottom of the style. The same is true for a certain L. mutabilis accession. - Abnormal penetration of the pollen tube in the ovule. Although penetration of the ovule was observed in L. unicolor Jacq. accessions a number of these penetrations were abnormal. - Embryo abortion and resulting non-viable seeds (Table 1). Initial attempts at embryo rescue in Lachenalia were not successful because of a lack of basic research on the development of Lachenalia embryos. As already mentioned the creation of intra-species hybrids or genepools is utilized to overcome the crossability problems to some extent (Table 3). Pure accessions of L. rubida Jacq. and L. bulbifera can successfully be crossed, but crosses usually result in very few viable seeds. Making intra-species crosses and then utilizing these intra-species hybrids to combine species gave better results (Table 3). Instead of less than 10 viable seeds per cross, the success rate was more than 50 viable seeds per cross which also resulted in more variation among the progeny.

CYTOGENETICS Lachenalia has a large variation in chromosome number. Basic chromosome numbers in the genus include x=5,6,7,8,10,11 and 13 and poliploidy is also present in several species (Moffett, 1936; De Wet, 1957; Nordenstam, 1982; Crosby, 1986; Ornduff and Watters, 1978; Hancke and Liebenberg, 1990; Kleynhans and Spies, 1999, Spies et al., 2000). The chromosome numbers of less than half, of the approximately 450 species accessions in the genebank at ARC-Roodeplaat, have been determined. Meiotic studies aid in the understanding of the relationship among the different species (Hancke and Liebenberg, 1998, Hancke et al., 2000, Du Preez et al., 2000).

CHANGING CULTIVATION PRACTICES Extensive cultivation research on Lachenalia has been done over the last 8 years. Cultivation practices are constantly being adapted and optimized for more effective production. These practices need to be incorporated in the hybrid evaluation process on a regular basis to ensure the selection of well-adapted cultivars. This presents problems as 235 some hybrids that were evaluated according to previous procedures needs to be re-evaluated. On the same level, problems with existing cultivars become evident as cultivation practices become more sophisticated. Existing cultivars thus need to be improved through breeding to comply with current cultivation practices.

DISEASE TOLERANCE AND RESISTANCE Except for the importance of the Ornithogalum mosaic virus (OMV) in Lachenalia little is known about the most important pests and diseases. As commercial cultivation increases pest and disease problems are constantly being encountered. These pests and diseases also need to be researched before breeding towards resistance can be attempted.

MUTATION BREEDING TECHNIQUES Due to the time constraints and compatibility problems in breeding new cultivars, it could be more profitable to make use of mutation breeding techniques to achieve certain objectives. Lachenalia is a good candidate for mutation breeding. Mutation breeding, however, will only be successful if the specific aims can not be obtained by making a single conventional cross. If more than one cross were required to obtain the desired result mutation breeding would definitely save time. The specific objective will thus determine if mutation breeding is a viable alternative.

Literature Cited Coertze, A.F., Hancke, F.L., Louw, E. and Niederwieser, J.G., 1992. A review of hybridisation and other research on Lachenalia in South Africa. Acta Hort. 325:605-609. Crosby, T.S. 1986. The genus Lachenalia. The Plantsman. 8(3):129-166. De Wet, J.M.J. 1957. Chromosome numbers in the Scilleae. 22:145-159. Du Preez, J.L., Spies, J.J. and Kleynhans, R. 2000 A preliminary study of interspecific hybrids in Lachenalia (Hyacinthaceae). V111th International symposium on flower bulbs. August 2000 Ferreira, D.I. and Hancke, F.L. 1985. Indigenous flower bulbs of South Africa: A source of new genera and species for ornamental cultivation. Acta Hort. 177:405-410. Hancke, F.L. and Coertze, A.F., 1988. Four new Lachenalia hybrids with yellow flowers. HortScience. 23(5):923-924. Hancke, F.L. and Liebenberg, H. 1990. B-chromosomes in some Lachenalia species and hybrids. S. Afr. J. Bot. 56(6):659-664. Hancke, F. L. and Liebenberg, H. 1998. Meiotic studies of interspecific Lachenalia hybrids and their parents. S. Afr. J. Bot. 64:250-255. Hancke, F. L. Jansen van Rensburg W.S. and Liebenberg, H. 2000. Chromosome associations of three interspecific, dibasic Lachenalia hybrids. S. Afr. J. Bot. (In Press). Kleynhans, R. and Spies, J.J. 1999. Chromosome number and morphological variation in Lachenalia bulbifera (Hyacinthaceae) S. Afr. J. Bot. 65:357-360. Kleynhans, R. and Spies, J.J. 2000. Genetic variation in Lachenalia bulbifera (Hyacinthaceae). Euphytica. 115:141-147. Lubbinge, J. 1980. Lachenalia breeding. I. Introduction. Acta Hort. 109:289-295. Lubbinge, J., Ferreira, D.I. and Van der Laarse, G.J. 1983a. The first red flowering Lachenalia cultivar, Rosabeth (ZA 81121). Agroplantae. 15:35. Lubbinge, J., Ferreira, D.I. and Van der Laarse, G.J. 1983b. Another Lachenalia cultivar, the orange flowering Roinge (ZA 81119). Agroplantae. 15:43. Lubbinge, J., Ferreira, D.I. and Van der Laarse, G.J. 1983c. A new pale yellow Lachenalia cultivar, Rolina (ZA 81118). Agroplantae. 15:39. Lubbinge, J., Ferreira, D.I. and Van der Laarse, G.J. 1983d. Lachenalia – the first purple flowering cultivar, Romargo (ZA 81122). Agroplantae. 15: 41. Malan, C.E., Ferreira, D.I. and Van der Laarse, G.J. 1983. Lachenalia - a new yellow flowering cultivar, Rodeas (ZA 81120). Agroplantae. 15:37. Moffett, A.H. 1936. The cytology of Lachenalia. Cytologia. 7:490-498. Nordenstam, B. 1982. Chromosome numbers of South African plants: 2. Jl. S. Afr. Bot. 48(2):273-275. Ornduff, R. and Watters, P.J. 1978. Chromosome numbers in Lachenalia. Jl. S. Afr. Bot. 44 236 (4):387-390. Spies, J.J., Du Preez, J.L., Minnaar, A. and Kleynhans, R. 2000. Hyacinthaceae: Chromosome studies on African plants. 13. Lachenalia mutabilis, L. pustulata and L. unicolor. Bothalia 30:106-111. Spies, J.J. Van Rooyen, P. and Kleynhans, R. 2000. The subgeneric delimitation of Lachenalia (Hyacinthaceae). VIIIth International symposium on flower bulbs. August 2000 Van Rooyen, P., Spies, J.J. and Kleynhans, R. 2000. The species delimitation of Lachenalia unifolia and L. hirta. VIIIth International symposium on flower bulbs. August 2000

237 Tables Table 1. The number of successful (O) and failed (X) F1 crossing combinations between some of the Lachenalia species accessions in the genebank at ARC-Roodeplaat. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. aloides bulbifera carnosa comptonii elegans framesii liliflora mutabilis namaquensis orchioides orthopetala pallida rubida splendida unicolor viridiflora L. aloides X=24 X=10 X=1 X=2 X=2 X=2 X=4 X=5 X=1 X=2 O=106 O=2 O=16 L. bulbifera X=2* O=17 X=2 X=13 X=8 O=19 X=3 X=5 O=8 S=1 O=2 L. carnosa X=1 X=2 X=1 X=1 X=1 O=2 X=1 O=2 X=1 X=1 O=3 O=1 O=2 O=2 L. comptonii X=3 X=1 X=1 X=4 L. elegans X=1 X=3 X=1 O=3 X=3 X=5 X=1 X=2 L. framesii X=1 O=2 O=1 X=1 L. liliflora X=1 X=1 X=1 L. mutabilis X=2 X=4 O=1 X=1 X=4 X=1 X=1 X=3 X=1 X=4 O=1 O=7 O=2 O=1 L. namaquensis X=3 X=3 O=2 O=1 L. orchioides X=2 X=1 X=1 X=3 O=10 X=1 X=13 X=1 X=2 O=6 O=8 O=2 L. orthopetala X=2 X=1 X=1 X=2 X=1 L. pallida X=1 X=2 O=1 X=1 X=1 X=1 O=1 O=1 L. rubida X=4 X=8 X=4 X=2 X=4 X=4 X=1 X=11 X=4 O=20 X=6 X=4 O=1 O=4 O=2 O=1 L. splendida X=2 X=5 O=3 X=1 X=1 O=1 O=1 X=3 O=5 X=1 O=2 O=3 O=2 O=2 O=1 O=2 L. unicolor X=10 O=2 X=1 X=1 O=1 X=3 X=1 X=1 O=2 L. viridiflora O=2 X=5 X=1 O=1 X=1 X=2 O=4 0=1 0=1 * Where the results of more than one F1 combination between two species are given these crosses were made between different pure species accessions of the same two species. For example different species accessions of L. bulbifera and L. aloides were used to make the 10 crosses between the two species. Eight were successful and two failed.

238 Table 2: Results of seed set after self-pollination of different Lachenalia species accessions.

Species Number of Number of accessions where Number of accessions where accessions with only 1-5 seeds per inflorescence seed set was obtained (more no seedset were obtained than 50 seeds) L. algoensis 2 L. aloides 32 4 L. arbuthnotiae 1 L. bachmanii 4 L. bulbifera 34 7 2 L. capensis 2 1 1 L. carnosa 12 3 1 L. comptonii 2 L. concordiana 5 1 L. congesta 1 2 L. contaminata 4 1 2 L. elegans 9 2 L. fistulosa 7 2 1 L. framesii 2 L. haarlemensis 2 L. hirta 6 3 L. isopetala 1 L. juncifolia 1 L. klinghardtiana 1 1 L. latifolia 1 L. liliflora 3 L. longibracteata 1 L. mathewsii 1 1 1 L. mediana 4 1 L. minima 1 L. mutabilis 12 10 9 L. namaquensis 6 1 L. orchioides 11 4 2 L. orthopetala 4 L. pallida 4 1 2 L. patula 1 L. peersii 2 L. pusilla 3 L. pustulata 10 9 2 L. reflexa 6 L. rubida 3 5 11 L. splendida 5 1 L. trichophylla 1 L. undulata 7 1 L. unicolor 15 14 11 L. unifolia 17 6 3 L. variegata 1 L. violaceae 9 11 L. viridiflora 4 L. zeyheri 1 Total 245 95 61

239 Table 3: Differences obtained after using an intra-species hybrid of L. rubida instead of pure accessions of the species in crosses with L. bulbifera.

Crossing Female parent and accession Male parent and accession number Result number number 1992/027 L. bulbifera (T1991/070) L. rubida (L174) No seed set and less than 10 non-viable seeds 1992/028 L. bulbifera (T1991/070) L. rubida (L379) No seed set and less than 10 non-viable seeds 1992/030 L. bulbifera(T1991/049) L. rubida (L174) Less than 10 viable and less than 10 non-viable seeds 1992/033 L. bulbifera (T1991/059) L. rubida (L277) Less than 10 viable 1993/098 L. bulbifera (T1991/059) L. rubida (L276) No seed set 1992/035 L. bulbifera (T1991/069) L. rubida (L174) No seed set 1992/039 L. bulbifera (T1991/064) L. rubida (L277) Less than 10 viable 1993/062 L. bulbifera (L389) L. rubida (L276) More than 50 viable seeds and less than 10 non-viable seeds 1993/063 L. bulbifera (L389) L. rubida (L277) Less than 10 viable and less than 10 non-viable seeds 1994/073 L. bulbifera (T1991/074) L. rubida intra-species hybrid (L174 X L277) More than 50 viable seeds and less than 10 non-viable seeds 1994/074 L. bulbifera (T1991/074) L. rubida intra-species hybrid (L174 X L381) More than 50 viable seeds and less than 10 non-viable seeds 1994/075 L. bulbifera (T1991/075) L. rubida intra-species hybrid (L276 X L379) More than 50 viable seeds and less than 10 non-viable seeds 1994/076 L. bulbifera (T1991/075) L. rubida intra-species hybrid (L174 X L276) More than 50 viable seeds and less than 10 non-viable seeds 1994/077 L. bulbifera (T1991/075) L. rubida intra-species hybrid (L174 X L277) More than 50 viable seeds and less than 10 non-viable seeds 1994/078 L. bulbifera (T1991/075) L. rubida intra-species hybrid (L174 X L381) More than 50 viable seeds and less than 10 non-viable seeds

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