South African Journal of Botany 2004, 70(1): 47–51Printed in South Africa — All rights reserved

Copyright © NISC Pty LtdSOUTH AFRICAN JOURNAL

OF BOTANYEISSN 1727–9321

Role of biotechnology in the development and production of Lachenaliaand Ornithogalum cultivars in South Africa

JG Niederwieser

Agricultural Research Council, Roodeplaat Vegetable and Ornamental Plants Institute, Private Bag X293, Pretoria 0001,South Africae-mail: fienie@vopi.agric.za

Received 2 September 2003, accepted 15 October 2003

This publication gives an overview of the role ofbiotechnology in the development and production oftwo indigenous flower bulbs in South Africa, namelyLachenalia and Ornithogalum. During the 1970s the twomajor constraints to successful commercialisation wereidentified as slow propagation rate and susceptibility tovirus, and work to overcome these was started. Later,more advanced techniques, viz. molecular markers,embryo rescue and in vitro bulb production, were devel-oped. Techniques such as virus elimination, mass prop-

agation, in vitro long-term storage, in vitro bulb forma-tion, embryo rescue and in vitro polyploidisation arebeing applied on a routine basis to shorten the timerequired to develop new cultivars, to maintain the prop-agation scheme and overcome incompatibility betweenspecies. More advanced technology gene transfer andmarker-assisted hybrid selection, may be applied infuture to develop cultivars resistant to virus and toincrease efficiency of hybrid selection.

In the 16th and 17th centuries, part of an exploration trav-eller’s mandate was to collect genetic material in newcolonies and transport it back to Europe where the plantmaterial was evaluated and those with economic potentialcommercialised. Botanists such as Hartman, Oldenland,Thunberg and Masson collected bulbous plant such asGladiolus, Freesia, Ixia, Lachenalia (Jacq.f. ex Murray),Zanthedeschia and Ornithogalum L. in the Cape in the 17th

century. These collections formed the basis of horticulturalenterprise development from South African plants (Bryan1989).

Of the genera mentioned above, Gladiolus and Freesiahave been developed in Europe and elsewhere into com-monly known multi-million dollar cut flower industries.Despite the fact that Zanthedeschia, Lachenalia andOrnithogalum possess characteristics such as goodvase/shelf-life, long stems and colour variation, develop-ment lagged behind and they were not developed to their fullpotential. Renewed interest in these genera developed afterit became clear that biotechnology can play a role in thedevelopment and commercialisation of these indigenousspecies. This paper focuses on the role of biotechnology todevelop Lachenalia (Figure 1) and Ornithogalum (Figure 2)in South Africa.

Ornithogalum thyrsoides Jacq. was the first fresh cutflower to be exported from South Africa. Flowers were col-lected from the natural habitat and exported by ship to the

United Kingdom in 1880 (Lighton 1960). Quality declined,however, possibly as a result of the spread of virus throughthe harvesting practices and today all cut flowers of O. thyr-soides are grown outside the natural habitat, for exampleIsrael, The Netherlands and the summer rainfall areas ofSouth Africa. Currently, more than 40 million stems are soldon the VBA (Dutch Flower Auction) and more than one mil-lion stems are sold at the Multiflora Flower Auction inJohannesburg annually. An estimated 20 million cut flowersare exported from South Africa annually.

In the 1970s, at least two institutions in South Africa, theRoodeplaat Vegetable and Ornamental Plant Institute and aprivate enterprise, initiated breeding programmes to com-bine the good cut flower characteristics (long vase-life andlong, straight stems) of O. thyrsoides with the yellow andorange flower colour of O. dubium Houtt. and O. maculatumJacq. Both programmes succeeded in obtaining yellow cutflowers, but by 1990 none of these hybrids were commer-cialised due to susceptibility of the genus to infection byOrnithogalum Mosaic Virus (OMV) (Burger and VonWechmar 1988). The problem with susceptibility to OMVwas not unique to the breeding programme in South Africaas Griesbach et al. (1993) and Wanghai and Bock (1996)reported the same problem elsewhere.

Fascination with the genus Lachenalia was evident soonafter colonisation of the Cape of Good Hope in 1652(Duncan 1988). Simon van der Stel used a painting of L.

Background

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hirta to illustrate the diary used during his expedition toNamaqualand in 1685. Since then, many collections ofLachenalia species have been established at botanical gar-dens, and by private enthusiasts and hybridisers. The bestknown hybrid originated in New Zealand and is erroneouslyknown as L. pearsonii. In 1985 a questionnaire by theIndigenous Bulb Growers Association of South Africa with itsworld-wide membership, revealed that the genus Lachenaliacame second only to Gladiolus in popularity. Despite theapparent popularity of the genus amongst bulb enthusiastsworld-wide, Lachenalia species and hybrids were not devel-oped into a commercial crop until late in the 20th century.

The Roodeplaat Vegetable and Ornamental Plant Instituteinitiated a breeding programme on Lachenalia early in the1970s. The objectives were to develop cultivars suitable forpot plant production in countries in the northern Hemisphere(Niederwieser et al. 1998). Bulbs were to be produced inSouth Africa and the dry bulbs exported. The first interestinghybrids produced in the early 1970s were discarded as aresult of severe virus infection. Thus, the first major problemto overcome during the early stages was to obtain virus-freenuclear plants of promising selections. The second was todevelop methods to mass propagate the virus-free plants.

Identification of the most limiting problem for commercial-isation, namely virus infection of Lachenalia andOrnithogalum cultivars during the 1970s was coincidentalwith the period when the potential of mass propagation and

virus elimination through tissue culture was realised by sci-entists and horticulturists throughout the world. Flower bulbsproved to be particularly amenable to tissue culture (Hussey1975). Development of techniques to free hybrid selectionsfrom viruses and to mass propagate them, received consid-erable attention at the institute from the late 1970s andthroughout the 1980s. The pioneering study by the institutewas that of Klesser and Nel (1976).

Another factor contributing to the extensive use of biotech-nology in the flower bulb programme at Roodeplaat is thetime required to develop and release new cultivars throughconventional breeding and propagation. The breeding cycleof Lachenalia is three years (Kleynhans and Hancke 2002),and for Ornithogalum two. In addition, the production cycle(production of a marketable size bulb starting with small bul-blets), is at least two years. Unless techniques are used tospeed up the propagation first of promising hybrids for large-scale evaluation, and second of commercial cultivars, devel-opment and release of new cultivars would require 18–20years.

Virus Elimination and Virus-free Nuclear Plants

Both Lachenalia and Ornithogalum are susceptible to OMV(Klesser and Nel 1976, Burger and Von Wechmar 1988).Infection results in decreased plant vigour, mosaic symp-toms on leaves and malformation of flowers. For Lachenalia

Figure 1: Red lachenalia: Lachenalia cultivar Rosabeth Figure 2: Yellow ornithogalum: interspecific Ornithogalum hybrid

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this is particularly critical as mosaic symptoms on leaves ofpotted plants are totally unacceptable to the market. Duringthe 1970s and early 1980s the conventional virus eliminationtechnique of combining heat therapy with meristem culturewas unsuccessful for Lachenalia and Ornithogalum. Vcelaret al. (1992) developed an improved technique by culturingleaf explants, excising adventitious buds and treatment withan anti-viral chemical.

Long Term Storage of Tissue in vitro

Maintenance of virus-free stock plants of Lachenalia andOrnithogalum is essential for sustainable production.Currently the institute maintains stock plants in insect-freegreenhouses as well as an in vitro backup collection of allcommercial cultivars using the method developed by Louw(1995).

Mass Propagation

Mass propagation by means of tissue culture plays a majorrole in flower bulb production throughout the world today.This is true also for Lachenalia and Ornithogalum in SouthAfrica. The slow natural propagation of most commercial cul-tivars and their susceptibility to OMV, make in vitro propaga-tion of ‘virus-free’ plants imperative to sustain production. Invitro propagation is used to obtain a sufficient number ofplants of promising hybrids for evaluation and once a selec-tion has been identified as a new commercial cultivar, massproduction is started. With the use of propagation in vitro thetime required to breed and release a new Lachenalia culti-var is 13 years instead of 18–20 years. Production ofLachenalia and Ornithogalum require a production schemebased on limited generation propagation and isolation ofmaterial of different phases within this scheme. Annual prop-agation of virus-tested nuclear plants by means of tissue cul-ture is essential to maintain the quality.

Extensive studies were conducted on adventitious bud for-mation and the factors affecting the process forOrnithogalum (Nel 1981, Landby and Niederwieser 1989)and Lachenalia (Nel 1983, Van Rensburg and Vcelar 1989,Niederwieser and Vcelar 1990, Niederwieser and VanStaden 1990a, 1990b, Niederwieser and Van Staden 1992,Niederwieser et al. 1992, Niederwieser and Ndou 2002).The totipotency of species and clones of both genera varyconsiderably and the physiological stage of the donor plantand the age of the leaf tissue play a major role in the expres-sion of the totipotency of the tissue. Constituents of the cul-ture medium play a secondary role in the extent of adventi-tious bud formation and the metabolism of plant growth reg-ulators appear to be determined by the genotype and tissueage.

As a result of the knowledge generated, tissue culture isapplied on a routine basis in the programme.

Advanced Breeding Techniques

Incompatibility between Ornithogalum species prompted astudy by Niederwieser et al. (1990) on embryo rescue toovercome incompatibility in cases where embryo develop-

ment is halted after fertilisation. By using a clearing-squashtechnique, it can be ascertained within 5h whether hybridembryos have been formed. Such embryos can be rescuedby culturing them in ovulo on basal medium containing 70gsucrose l–1 and no added growth regulators. The embryos’requirement for sucrose changes as they develop. Thereforecultured ovules are transferred after 14 days to a mediumcontaining 10g sucrose l–1, where germination occurs. Thistechnique was later applied at the institute not only to rescueembryos of incompatible crosses, but also as a way to short-en the two- to three-year breeding cycle. Embryo rescue hasnot been applied successfully to Lachenalia until now.However, it is becoming evident that intra- and inter-speciesincompatibility is limiting progress in the breeding pro-gramme. Of all the failed crosses made at Roodeplaat, 44%failed as a result of non-viable seed (Kleynhans and Hancke2002). Initial attempts to rescue embryos using the tech-nique for Ornithogalum failed. Clearly, the technique usedfor Ornithogalum should be adapted for Lachenalia to over-come incompatibility.

Incompatibility between O. dubium and O. thyrsoides maybe overcome by polyploidisation of sterile F1 hybrids(Blomerus 2002). Preliminary results by Blomerus indicatedthat in vitro colchicine treatment can be very successful toobtain tetraploid plants. Leaf explants with clearly visibleadventitious buds were treated with 0.2% colchicine for 48h.Using the in vitro approach, the colchicine had easy accessto the meristematic area and 54–100% of the regeneratedplants were tetraploid. Increased ploidy levels in Lachenaliado not result in increased vigour or bigger flowers, but workis underway to overcome incompatibility between specieswith different chromosome numbers through polyploidisationusing tissue in vitro (Kleynhans pers. comm.).

Molecular Markers

Molecular markers have been used to study intra-speciesvariation of L. bulbifera (Cyrillo) Engl. (Kleynhans and Spies2000). If important plant or cultivation characteristics can belinked to specific molecular markers, this technique has thepotential to increase efficiency of hybrid selection. However,this will require substantial study in the future.

Gene Transfer

Although control of virus infection is accomplished throughapplication of a limited generation propagation scheme forboth Lachenalia and Ornithogalum cultivars, it is an expen-sive and management-intensive procedure. Resistance tovirus infection would be the ultimate solution and will reducethe production cost considerably.

The study of Burger and Von Wechmar (1988) to identifyand characterise Ornithogalum Mosaic Virus (OMV), formedthe basis for research to transfer resistance to OMV intoOrnithogalum and Lachenalia cultivars. De Villiers (1999)and De Villiers and Bornman (1999) developed a tissue cul-ture system that was suitable for transformation studies andoptimised a transient transformation protocol with the GUSgene. Stable transformation was attempted in two laborato-ries using the pat herbicide resistance gene. Putative trans-

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genic plants obtained in the laboratories of the University ofCape Town, were subjected to molecular analysis andclones derived from one plant were found to be resistant toherbicide and greenhouse trials indicated that plants werephenotypically normal and that leaves re-introduced into tis-sue culture could regenerate plants. Thus the scene was setand work to transfer the susceptibility to OMV to hybridscould commence. Progress has been slow but steady duringthe past few years. Recently researchers at Roodeplaatobtained a number of putative transgenic plants and the sys-tem will be optimised to increase the number of these plants.

Physiological Studies

As with most other bulbs, temperature plays a major role inthe growth and development of Lachenalia. Louw (1993) stud-ied the effects of temperature during the bulb dormant phaseon inflorescence development. Du Toit (2001) showed thattemperature during the bulb preparation phase (the seasonbefore bulbs are potted or forced) affects the formation ofdaughter bulbs, inflorescence development during the dor-mant phase and plant morphology during forcing.Unpublished results of current trials at Roodeplaat indicatethat temperature just prior to and during forcing has a signifi-cant effect on the period from planting to flower, the numberof flowers per inflorescence and uniformity of flowering date.Progress of this research is hampered by the fact that expen-sive climate controlled greenhouses or phytotrons arerequired. Slabbert and Niederwieser (1999) showed that invitro bulb formation by shoots regenerated from leaf tissue ispossible through manipulation of temperature and sucroseconcentration. By using this system, plants of uniform sizeand a known history can be subjected to a number of tem-perature treatments at a fraction of the cost of greenhouse tri-als. In vitro studies to obtain basic information on the effect oftemperature on different growth stages are currently under-way.

Conclusion

Application of biotechnology made it possible to develop andcommercialise two indigenous flower bulb genera, namelyLachenalia and Ornithogalum. These techniques includevirus elimination, mass propagation, in vitro long-term stor-age, embryo rescue and in vitro application of mutagens toobtain polyploidy. Information pertaining to basic aspectsincluding the origin of adventitious buds on leaf tissue,regeneration pathways, variation within Lachenalia species,relationships between tissues in leaves and effects of hor-mones, tissue age and culture medium components onadventitious bud formation, as well as cytokinin metabolismwas generated. Advanced techniques such as gene transferproved to be more difficult but progress on Ornithogalumhas been made over the years and may prove to be veryvaluable in the future to overcome the biggest constraint tosustainable production, namely virus susceptibility.

Acknowledgements — Thanks are due to the Agrcultural ResearchCouncil for financial assistance and to Chris Bornman for the oppor-tunity to write this review.

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Edited by CH Bornman