S. Afr. J. Bot. 1999. 65(5 & 6): 361 - 369 361
Micropropagation of members of the Hyacinthaceae with medicinal and ornamental potential- A review
S.A. McCartan and J. van Staden* Research Centre for Plant Growth and Development, School of Botany and Zoology, Un1vers1ty of Natal P1etermaritzburg, Private Bag X01 , Scottsville, 3209 Republic of South Africa
Recetved 5 May I')')!); revtsed I September 19!.19
The Hyac1nthaceae comprises many genera, which are characterized by bulbs, thick roots, basal leaves and simple or more rarely branched racemes. These genera are widely exploited for their medicinal, pharmaceutical and ornamental potential. In South Africa, these plants are harvested without permits from wild populations, processed and then sold as traditional medicine. This is having a negative impact on wild populations. Although the active ingredients of many of the bulbs have not been identified, several bufadienolides such as proscillaren A have been isolated from members of this family. These bufadienolides have pharmaceutical potential as cardiotonics. Thus, many members of the Hyacmthaceae, have potential as alternative horticultural/agricultural bulb and/or flower crops for medicinal, pharmaceutical and ornamental purposes. Conventional propagation of these plants, however, is usually fairly slow. M1cropropagalion provides a rapid means to propagate selected chemotypes or cultivars, servmg both conservation and commercial interests. Many members of Hyacinthaceae have been micropropagated This review summarizes th1s information, highlighting the potent1al and problems surrounding this family of flowering plants.
Keywords: adventitious shoots, axillary shoots, bufadienolides, conservation, in vitro propagation, somatic embryos, tissue culture.
Abbreviations: 2,4-D = 2,4-dichlorophenoxyacetic acid; AC = activated charcoal; 85 = Gamborg medium: BA = benzyladenin e, BDS = modified 85 medium: CH = casein hydrosyla te: CM = coconut m1lk; IM = indole-3-acetiC ac1d: IBA = indole-3-butyric acid; iP = 6-[y,y-dimethylallylamino]purine: kinetin = N6-furfuryladenine; MS = Murashige & Skoog medium: NM = •x-napthyaleneacetic acid; PAC = paclobutrazol; TDZ = thidiazuron: YE = yeast extract; (3G]BA, [7G]BA, (9G]BA = 3,7,9 glucosides of benzyladenine.
• To whom correspondence should be addressed.
Introduction Wyk eta/ 1997). The Asparngales forms a large and fairly homogenous complex $'cilia natalensis is one of the top-ten most popular med icinal of fami lies. It is believed to be a monophyletic group, which plants in South Africa (Cunningham 1988; Williams 1996; Man evolved in parallel with the Liliales and Dioscoralcs. The Aspar der 1997). Despite its specially protected conse rvation status. agales consists of 31 families including several new families. approximately 95 tons of these bulbs are sold illegally in Durban These fami lies. which include the Alliaceae, Asparagaceae, annually. The price of these bulbs is relatively low ranging from Asphodelacene, Dracennaceae, Eriosperm aceae and Hya R 1.89 to R6.80 per kilogram. This is mainly due to the occur cinthaceae. comprise genera previously placed within the Lil rence of large populations of these bulbs locally. T he price of another popular medicinal plant, Bowiea volubilis is substan iaceae Wll.\'11 fato (Dahlgren et a/. 1985; Perry 1985). The Hyac inthaccae comprises several ge nera including Bo111iea. tially higher ranging from R 11.74 to R27.80 per kilogram (Mander 1997). The price of these bulbs has increased steadily F.u~·omis, Lachenalia, Ledebouria, Scilla and l/rginea. These with the decline in their availability and size (Cunningham genera arc charncterised by bul bs with thick and sometimes con 1988). tractile roots. basa l leaves and simple or more rarely branched Although the active ingredients of many of these bulbs have racemes. The inflorescences comprise actinomorphic, bisex ual not yet been identified. several bu fadienolides have been isolated tlowers, which range from white, blue, violet, and more rarel y from members of the Hyacinthaceae including Bml'iea I'Oiubilis yellow, red, brown to nearly black. T he Hyac inthaceae are (Jha 1988; Finnie et a/. 1994), Drimia rohu.1·ta (van Wyk eta/ widely distributed, occurring in southern Africa and in a region 1997; Luyt et a/. 1999}, Urginea a/tissima (Hutchings et a/. from tht: Mediterranean to SW As ia (Dahlgren et af. 1985; 1996), U. indica (Jha & Sen 198 1, 1983; Jha 1988) and (} mar Duncan & du Pless is 1989). itima (Shyr & Staba 1976; Verbiscar eta/. 1986; Gentry el a/ 1987; Jha 1988). Bufadienolides such as proscillaridin A are val Medicinal and pharmaceutical potential uable since they can be administered to digital in sensitive/intol In South A frica, approximately 20 000 tons of plant material, erant patients (Jha 1988). These bufadienolidcs. however. are worth about R270 million, is harvested, processed and so ld as usually not used as cardiotonics due to their low therapeutic indi traditional medicine annually (Gosling 1998 }. Approximately ces (Jager & van Staden 1995). However, some bufadienolides 14% of this plant material comprises bulbs (Mander 1997}, identified in certain organs of Bowiea l'oluhi/is are thirty to sixty which are destruct ive ly harvested, processed and so ld for th e times more active than those fi·om Digitalis (Hutchings et a/ treatment of various ai lments (Table I ). These bulbs, which are 1996). Prosc illaridin A, which is derived by enzyme hydrolys is sold whole, sliced or chopped, are usually administered as decoc from scillaren A , is produced under several trade names includ tions, emetics and enemas (Watt & Breyer-Brandwij k 1962; ing Caradin®, Cardion®, Prosc illan®, Sandosc ill®, Scillac Jacot Guillamod 1971; Hutchings 1989; Mander et af. 1995; van rist®, Talusin® and Urgilan® (Budavari 1996). T hese cardiac 362 S. Afr. J. Bot. 1999, 65(5 & 6)
Table 1 Medicinal uses of members of the Hyacinthaceae in South Africa Plant namc 1 Medicinal uses References Alhuw mnadensis (L) Leighton syn. A. As an anthelmintic, thirst quencher anc.l to treat Watt & Brcyer-Brandwijk I962; Hutchings JllllfOI" venereal c.liseases 1989 Albuca coopen Bak. As an anthelmintic, lotion for washing wounds and Watt & Breyer-Brandwijk 1962; llutchings to treat venereal diseases 1989 .·1/huca fusugaw ( L.f.) Dryanc.l. To treat illnesses caused by poisons Hutchings eta/ 1996 Alhuca selosa Jacq. As an anthelmintic, lotion for washing wounds in Jacot Guillamnd 1971; Hutchings 19R9 animals and to tn:at venereal diseases A/hum slwH"ii Bak. syn . ..1. trichophylla As an anthelmintic and to treat constipation and Walt & Breycr-Brandwijk 1962; Jacot Guil gonorrhoea lamnd 1971; Hutchings 1989 Bml"iea m/uhilis Harv. To treat c.lropsy. barrenness in women and head Walt & 13rcycr-l3rantlwijk 1962; Batten & aches Bokclmann 1966; Hutchings 1989; Hutch ings eta/ 1996 /Jtpcatli hreviofo/ium (Thunb.) Fnurc. To treat heart pains and breathlessness 11utchings 1989; Hutchings et al 1996
fJtpcatlt gmcillimum Bak. syn. /J. po~vphyf- To treat gonorrhoea and pimples Watt & 13rcycr-13randwijk 1962 fum Oipcaclt \'ll"tde (L.) Moench To treat gonorrhoea and flatulence Watt & Breycr-Brandwijk 1962; l3attcn & Bokelmann 1966: Hutchings 1989; llutch ings et a/ 1996 !Jrimia ctliaris Jacq. As an emetic. expectorant and diuretic Walt & Breyer-Brandwijk 1962 f)rimia data Jacq. To treat high blond pressure and stabbing pains Hutchings el af 1996 /)nmiallem!formi.l· Bak. To treat extemal tumours that have been lanced Watt & Breyer-Brandwijk 1962; Jacot Guil lamod 1971
f)rimia mhusta Oak. syn. D. alw As an expectorant, emetic and enema to treat Hutchings 1989; Hutchings et af 1996 feveri sh colds Ez""OIII/ .1" 1111111111/Utlis (Mill.) Chill. To treat co lic. llatulence, syphili s and hangovers Hutchings et af 1996 f:"ummts hicolor Bak. For colic and as a purgative Watt & Breyer-Brandwijk 1962; Batten & 13okclmann 1966; Hutchings et af 1996 f:'ucomts mmo.w (Houtl.) var. comosa syn E. Tn treat rheumatism and teething infants Walt & Brcyer-Brandwijk 1962; Batten & JIIIIIC/(1/(1 l3okclmann 1966; llutchings 1989: Hutch ings eta/ 1996 t:ucomts JIO/eevansii N .E. Br. For people sut1'ering from mental disease Wall & Brcycr-Brandwijk 1962 f:"ucoll/1.1" regia (L.) L 'Heril To treat venereal disease, lumbago, diarrhoea, Watt & l3rcycr-Brandwijk 1962 respiratory conditions especially cough nnd biliousness and to prevent premature childbirth f.l!clehrwria cooperi (I look. I:) Jessop syn. Used in the initiation ceremony of boys; to treat Watt & Brcyer-Brandwijk 1962; Hallen & Scilla cooperi. S. immdensis. S. satum gastro-intestinal, gynaecological and psychologi !3okclmann I 966: Hutchings 1989: Hutch cal ailments ings et a/ 1996 f.edehmll·w ovatifolia (Oak.) Jessop syn. To treat gastro-intestinal and gynaecological llutchings 1989; Hutchings et of 1996 Scilla m·wifolia ailments, backache and inllucnza l.edehouno rewJ!wa (I.. f.) Jessop syn. Scilla For treating lumbago and gall sickness in animals; Watt & Brcyer-Brandwijk 1962: Jacot Guil faiiCI!III!(Ofill to bathe skin eruptions anti as an ointment for lamod 197 1 wounds and sores .\/a.uonut echinata L.f. syn M . bowker1 ror ophthalmic applications. sterility and tooth Walt & Breyer-Brandwijk 1962; Jacot Guil ache h:mod 1971 Ormtlwgalum longl/Jractealllm Jacq. To treat swellings or growths Hutch ings 1989: Hutchings et a/ 1996 Ormtlwgalum duhiwn Houtt. syn 0. mima- As an anthelmintic BaHcn & Bokel mann 1966 ll/111 Ormtlwgalum lr!lllljolium De Iaroche subsp. As a rat poison Watt & Brcyer-Brandwijk 1962: Jacot Guil tenui(ofiunz syn. 0. eckfonii & 0. viren.1· lamod 197 1 Ormthoj Table 1 Continued /.'rgineaaltJssima (I.. f.) Bak. To treat rheumatic swellings, gastro-intestinal and Watt & 13rcycr-Brandwijk 1962; Hutchings respiratory ailments 1989; Hutchings eta/ 1996 f irginea e/11gea R.A. Dyer As a treatment f()r colds anti headaches Wall & Rrcycr-Brantlwijk 1962 l :rginea mciC-roce/ltra Bak. As an anthelmintic Hutchings eta! 1996 I "rgmea phy.mdes (Jacq.) Bak. To treat gynaecological ailments and to facilitate Hutchings I989: llutchings eta/ 1996 delivery during birth l ."rgmea rubella Bak. For the treatment of colic Watt & Brc)cr-Brandwijk 1962: Jacot Cluil lamotll971 l r~;mea .wmgumea Schinz. syn. li. burkei As an abortifacient and lo treat paralysis, circulatory Watt & 13rcycr-Brantlwijk 1962 diseases and rheumatism pains ' Plant name and synonyms (Amok! & De Wet 1993). glycosides increase the force of the systolic contraction, inhibit Conventional propagation lhe atrioventricular conduction, and enhance the 'automacy' of Several members of the Hyacinthaceae, therefore, have potential the cardiac tissue and. therefore, are used to treat heart fai lure as alternative horticultural/agricultural crops, providing crops of and certain arrhythmias (Walton eta/. 1994). both bulbs and inflorescences. Seed, however, is seldom used for 13ufadienolides such as scill iroside and scillaren A also are commercial propagation, except where the species or cultivars effective rodenticides with the French Pharmacopoeia Codex produce large quantities of reliable seed with short juvenile peri describing a raticidal paste composed of powdered bulb of Red ods (Rees 1992). Some members of the Hyacinthaceae including Squill ( LlrKinea maritima), fl our and sugar (Balbaa eta/. 1979). Scilla siberica, S. siberica cv •A lba', and S. bifolia, however, are The erticicncy of these strongly emetic rodenticides is largely only propagated by seed, while others including S. siberica cv due to the inability of rodents to regurgitate. This renders these 'Spring Beauty' and S. tubergeniana [= S mischtschenkoana products extremely safe and specific since humans and domesti c (Bryan 1989)] are propagated vegetatively (Le Nard & De Her animals including chickens, cats and dogs readily regurgitate tagh 1993). Although offsets are used to propagate many mem accidentally-i ngested baits (Crabtree 1947). In the 1940's, sev bers of the Hyacinthaceae, thi s is generally too slow for commercial propagation. Several artificial techniques, which eral tons of powdered bulb of Red Squill ( Urginea maritima) include scaling, basal cuttage (scooping and scoring) and bulb were imported into the USA. This ceased with the introduction cuttings, are used to increase the rate of natural mu ltiplication. of warfarin and other anticoagul ant-type rodenticides. The devel These, however, are usual ly restricted by the size of the mother opment of warfarin-resistance in rodents, however, has led to bulb. renewed interest in Red Squill-formulated products as co-roden ticides ( Vcrbi scar eta/. 1986). This also has led to the identifica Micropropagation tion and quantification of bufadienolides in various clones or Several members of the Hyacinthaceae have been micropropa cy totypes of Indian Squill (U indica) (Jha & Sen 198 1, 1983) gated using various techniques. These include the pro liferation and Red Squill ( li. maritima) (Gentry et a/ 1987) to select of axillary shoots, the ini tiation of adventitious shoots and the high-yielding strains suitabl e for commercial exploitation. induction of somatic embryos, which are discussed in greater detail (Table 2). Some details, however, such as the frequency of Ornamental potential shoot initiation, the average number of shoots initiated, the fre Approximately 16 000 ha are allotted to ornamental bulb produc quency of fasciated, deform ed or mutated shoots, the frequency tion in the Netherlands, representing 55% of the world 's total of plantlets successfully acclimatized, and the propagation production area, with signifi cantly smaller areas allotted to orna potential within a specified tim e-frame are seldom reported. mental bulb production in the USA (4 449 ha), the UK (4 300 These details, which should be included routinely in micropropa ha). Japan (I 622 ha), France ( 1 285 ha) and South Africa (425 gation reports, are essential for comm ercial fac ilities. hnl (De Hertogh & Le Nard 1993a). Desp ite the ornamental potential of the Hyacinthaceae. the area allotted to the production Axillary shoots of /~un1111is (2 ha), Galtonia (I ha), 1/yacint/ws (955 ha), Orni In Eucomis. axi ll ary shoots were proliferated from twin-scale tlwga/um (50 ha), Scilla (20 ha) and Urginea (3.2 ha) is small explants, whi ch were cultured on medium containing combina (De He11ogh & LeNard 1993 a, b; Le Nard & De Hertogh 1993 ), tions of BA and NAA. These shoots, which were then sub-cul represen ting approximately 3% of the world 's total ornamental tured onto the same medium, proliferated more shoots resulti ng in a continuous culture system. After 8- 10 weeks, the shoots bulb production area. Many of these genera including Hyacin were then rooted on medium with or without NAA (Ault 1995b). thus. Lachenalia and Scilla are worthwhile pot plants, while oth Although axillary shoots are genetically-stab le, the limited ers includ ing Eucomis. Ornitlwgalum and Urginea are useful availability of these axillary shoots restricts the potential of this garden plants (De Hertogh & Le Nard 1993b; Le Nard & De technique (Hussey 1980). Furthermore, 'mixed' cultures com Hertogh 1993 ). A breeding programme for Lachenalia compris prised of axillary and adventitious shoots do occur occasionally ing five phases including the establishment of a genebank, and (H ussey 1976a). The origin of these axillary shoots is seldom the development and subsequent evaluation of hybrids for com confirmed, and thus, the frequency of 'mixed cultures' is rarely merciali zation has been established over the past thirty years reported. (N icderwieser eta/. 1998 ). The successfu l introducti on of these hybrids onto the international flower market could initiate other Direct adventitious shoots breeding programmes, increasing the popul arity of these and In several members of the Hyaci nthaceae, adventitious shoots other plants. were initiated directly on, or along the periphery of, various 364 S. Afr. J. Bot. !999. 65(5 & 6) Table 2 Micropropagation of some members of the Hyacinthaceae Plant name 1° and 2° Explants Medium an<.! supplcments 1 Growth response Refi:rences /Jowiea \'oluhilis Bulb sc /Jou iea mluhilis lntlorescence Mo<.litic<.l MS. Sucrose (30000}. CM ( 150). 2.4-D (I ). Ag <~r Callus .lim & Sen !9!15 (6000) Bulb-scales ~nd split Modi lied MS. Sucrose (30000). Cll ( 100). 2.4 -D (0.5), Shoots shoots Agar (6000) Modi tied MS. Sucrose (30000), Agar (6000) l'lantlets Shoots Modi fied MS. Sucrose (30000). BA (2), 2.4-D (0.05) l'lantlets Shoots Bowrca l'olubilts Twin-scales MS. Sucrnsc (30000). BA ( I0). NAA (I), Agar (8000) Shoots Van Staden e1 a/. (991 Orimia rollltsta 13ulb-scalcs MS. Sucrose (30000}, 13A (2), NAA ( 1). Ag<~r ( 10000) Bulblets Ngugi era/ I J:ucomi.~ att1wn1w- Twin-scales H·o m bulbs MS . Sucrose (30000), BA (0-5). NAA ( 1), Agar (7000) Shoots Aull 1995b li.~. [. comosa & £. Shoots MS . Sucrose (30000). NAA (0-2), Agt1r (7000) Plantlets =umhe.1·iaca Eucoll/1.\' aurw1ma- Leaves and bulb-scales MS. Sucrose (20000}, BA ( 1-2). NAA ( 1-2), Gelrit e (2000) Shoots Ta)lor& Van h I:· mnw.1·a. E. MS. Sucrose (20000). lAA ( 1-2) or I BA ( 1-2), Gelrite Stndcn 1998 lun>lor, E lmmilis. Shoots (2000) l'hmtlets E. =ambeswca (rigure lA) Eun mrt.1· polccmn- Seeds Modified MS, Sucrose (20000), Gdritc (2000) Secdl i n~: s McCartan & Van stt Leaves of seedli ngs Mndi lied MS . Sucrose (20000), NAA ( 1), Gelrite (2000) Shoots (Figure 1Bb) Staden I 995 Shoots Modified MS. Sucrose (20000). lAA ( 1), Gelr ite (2000) Planllcts Eucomt.l \'(}}lder- Leaves Modi fi ed MS. Sucrose (20000). 13A (1-2), lAA (I). Gelnte Shoots McCartan et of. 1111!1' \1'1..'1 Shoots (2000) Pluntlcts (In Press) Modi tied MS. Sucrose (20000). 113A (I), Gdrite (2000) ( ialtonio <'llllllt l'e<.liccls & ovary bas~s MS. Sucrose (30000). BA (0.3),NAA ( 1) , Agar (8000) Shoots Drewc:s & V;m mlls & (i l'trldr Split shoots MS (Y: ). Sucrose (30000). NAA (0.5). Agar (8000) Plantlcts Staden 1993 j/om lf.racttllhus orren- Leaves MS. Glucose or Sucrose (30000). 13A ( l ). NAA (0. 1) . ± 13 ulblcts Bach et a/. I <)')2 ta/1.1· cv. Delft's PAC (8), Ag<~r (8000) 131 u ~ I f.mcmtlm.t cmen- Basal parts nf bulbs Modi lied Knops (V.,). Glucose (20000). lAA ( 10). Agar 13ulblcts Pi erik & Stcc:g talis cv. Pink Pearl (6000) mans 1975 flyad mhu.1· sp cv. Twin-scales ti·om bulbs MS. Sucmsc (20000). Agar (7000) Shoots Hussc} 1975 Ostara & Princes Leaves. stems & ovary MS. Sucrose (20000). lA A (2-8) or NAA (0.03-0.12). Agar Shoots & callus lrcn..: Split shoots and callus (7000) Plantk ts MS. Sucrose (20000). Agar (7000) l.uchenolw Leaves MS. Sucrose (30000). 13A (2). K-NAA (0.1). Ag<~r (6000) Shoots Auh 19 Table 2 Continued lfmcari armenia- 13ulb scales Modi lied MS, Sucrose (30000). BA (5}, NAA (2}. AC Bulblets Peck & Cumming cw11 cv. Early ( I 000). Gelrite (2000} 1986 (jiant Ormthogalum cv. Leaves Modified MS, Sucrose (30000}, BA (2}, NAA (0.1 }, Agar Shoots Landby & Nicd Ro llmv (7000} erweiser 1992 Ornithor.alum cv. Leaves Modi tied MS. Sucrose (30000}, BA (2}, NAA (0.1) . Gel rite Buds Vcela r et a/ I 992 Rogel Meristems from buds (2000) Virus-indexed plant- Modilied MS, Sucrose (30000), BA (I}, NAA (I}, adenine lets arabinose ( I), Gelrite (2000} ()mithogalum Leaves Modi lied MS, Sucrose (30000), BA (2), NAA (0. 1} , Agar Shoots Nel 198 1 hybrid Shoots (7000} Plantlcts Mod ified MS. Sucrose (30000), Agar (7000} On1ithoga/unl Leaves Mod ified MS, Sucrose (30000). BA (2), NAA (0.1). Agar Callus & shoots Van Rcnsburg et IIIOCII/lllllll/ (7000} a!. 1989 ( Jmithogalwn Stem, Leaves. Ovary, MS. Sucrose (20000), Agar (7000) Plantlets Hussey 1976h tlm·.wides Sepals, Bulb scales Omithogalum Twin-scales of bulbs MS, Sucrose (30000),NAA (8}, Ag;~r (5000) Callus Nayak & Sen umhellatum Callus MS. Sucrose (30000), BA (0.5), NAA (2), Agar (5000} Shoots 1995 Shoots MS ('/>}, Sucrose (30000), Agar (5000) Plantlcts Sclli:ohmis ino·i Bulb Scales MS, Sucrose (30000), BA (2). NAA (2), Agar (8000) Shoots and callus Drewes eta/. 1 cctla Shoots MS ( /:), Sucrose (30000), NAA (I), Agar (8000) Plantlets 1993 Sed/a hyacinthi- Leaves MS, Sucrose (30000), Agar (I 0000) Shoots Nair 1989 ww Shoots MS. Sucrose (30000), Kinetin (5), NAA (I), Agar ( 10000) Shoots Shoots MS, Sucrose (20000) Plantlcts Scilla nawlensi.~ l3ulbs Modi tied MS. Sucrose (20000), Gelrite (2000) Shoots McCartan & Van ( Figun: I C) Leaf and bulb scales of Modi tied MS. Sucrose (20000), Kinetin ( 1-2), IAA ( 1-2), Shoots (figures I D Staden 1998 shoots Gelrile (2000) & E) Shoots Moditicd MS, Sucrose (20000). IAA (I), Gel rite (2000) Plantlets /lwmntho.f basuti- Bulb scales MS. Sucrose (30000), BA (0. 1), NAA (5), Agar (8000) Ptantlcts Jones eta/. 1992 Cl/111 / .'rgmea indica Twin-scales of bulbs Modi tied MS, Sucrose (30000), Kinetin ( 1-2), 2,4-D ( 1-4), Callus depending on Jha et a/. t 991 (2n. 3n 7 4n} Callus ± NAA (2), ± CM ( 150), ± YE (I000), Agar (6000} ploidy Callus Mndi fit:d MS. Sucrose (30000), Kinetin (2}, 2,4-D (0.5), ± Shoots depending NAA (0.5-1 ), ± CM ( 150), ± YE ( 1000), Agar (6000) on ploidy Modi tied MS. Sucrose (30000), Kinetin ( I) or 2,4-D ( I), Somatic embryos Agar (6000) depending on ploidy / .'rgtnea mdica Twin-scales of bulbs Moditkd MS. Sucrose (30000). 2,4-D (2), CM ( 150), Agar Callus and somotic Jha ct a/. 1986 Somatic embryos (6000) embryos Plantlets Modi tied MS, Sucrose (30000), BA (0.1 ), CM (I 00}. Agar Plantlets (6000) Bulbous plantlets Moditlcd MS. Sucrose (30000), Kinetin (0.05), NAA (0.01 ). Agar (6000} llrgmea indica Twin-scales of bulbs Modi tied MS , Sucrose (30000), 2,4-D (2), CM (150) OR, Callus and shoots .lha eta/. !984 Shoots Kinetin (2), NAA (2), YE ( 1000), Agar (6000) Plantlets MS ('h), Sucrose (5000), Agar (6000) Urginea maritima Bulb scales Modified MS, Sucrose (40000), BA (0.1-0.3), NAA (0. 1- Plantlets El Grnri & Back Bulblets 0.3 ), Agar (7500) Bulblets haus 1987 Modified MS, Sucrose (40000), NAA (0. 1-0.3), Agar Ptantlets (7500} l"cltheimia hracte- Leaves and nora! stems MS. Sucrose (30000). BA (2), NAA (0.1), Agar (7000) Shoots Ault 1996 Ilia cv. Lemon Shoots MS. Sucrose (30000), K-NAA (0-2), Agar (7000) Plnntlels Flame & Rosalba & /" capensis /"e/theimia bracte- Leaves and bulb-scales MS. Sucrose (30000}, BA (2), NAA (0.1 ), Agar (8000) Shoots (Figure I G) Taylor & Van ala (Figure t F) Shoots MS, Sucrose (30000), IBA (2), Agar (8000) Plantlets (Figure I H) Staden 1997 1Fig ures in parentheses indicate mg 1·1 or m11· 1• explants. T he explant source, orientation, position and size, how ieser 1992). In Scilla, the explant source influenced shoot initia ever, influenced shoot initiation (Niederwieser & Van Staden tion with leaf ex plants producing more shoots than bulb explants t 990a & 1992: Niederwieser &Vcelar 1990; Landby & Niederw- (McCartan & Van Staden 1998). In Lachenalia, the age of the :>li6 S. Afr. J. Bot. 1999, 65(5 & 6) Figure I :\: lntlorescences of Eucomis hybrids and species; B: Adventitious shoot initiated along the periphery of a leaf explant or E. poleevcmsii: C: lntlorescence of Scilla natalcnsis: D: Adventitious buds initiated on the adaxial surface of a leaf explant of S. natalcnsis; E: Adventitious shoots initiated on a leaf explant of S. natalcnsis; F: Inflorescence of Veltheimia bracteata cv. Lemon rtame; G : Plantlcts of V. hrat·leata cv. Lemon Flame H: Acclimatized plants of V bracteata cv. Lemon Flame. S. Afr. J. Bot. 1999, 65(5 & 6) 367 cxplants intluenced shoot initiati on, with young ex plants produc Callus and indirect adventitious shoots ing more shoots than old explants. This may have been linked to In Boll'iea, callus was initiated on medium containing 2.4-D and endogenous cytokini n levels particularl y in the intermediate and coconut milk, and then transferred to medium containing 2,4-D old explants (N iedcrwieser & Van Staden 1990a). In Ornithoga and casein hydrolysate. This callus was comprised of green nod lum, the orientation of the explants also influenced shoot initia ules, which produced adventitious shoots, when transferred to tion with <~p olar explants producing more shoots than polar med ium containing low concentrations of 2,4-D (Jha & Sen ex plants ( Landby & Niederwieser 1992), whi le in Lachenalia. 1985). In Omithogalum. callus was initiated on medium contain callus and deformed shoots were formed when explants were ing NAA alone (Hussey 1976b; Nayak & Sen 1995) or in combi cultured with the adaxial surface down (Niederwieser & Vcelar nation with BA (Van Rensburg et a!. 1989). The frequency and 1990). In l.ucllenalia, the explant size influenced shoot initiation size of the callus decreased with increasing sucrose concentra wi th small explants producing more shoots than large explants. tion (Van Rensburg eta/. 1989). Hussey (1 976b) found that the The optimum size explant was 3.3 x 15 nun {Niederwieser & call us was comprised of a mixture of diploid and tetraploid cells. Vcelar 1990). This may have been related to the wound surface, He al so fo und that the frequency of tetraploid ce ll s increased which is proportionally larger in smaller explants. In Omithoga with callus age (Hussey 1976b). In contrast, Nayak and Sen lum. however. additional wounding on the surface of the explant ( 1995) found that the callus was genetically-stable for several inhibited shoot initiation (Landby & Niederwieser 1992). In years. The callus produced adventitious shoots when transferred I ~mcimlws. th e explant size also in fluenced shoot initiation, to medium containing no plant growth regulators (Hussey which decreased linearly with a decrease in explant size (Pierik 1976b) or medium con taining combinations of BA and NAA & Post 1975). (Nayak & Sen 1995). In Urginea, callus was initiated on medi um These adventitious shoots originated from single cells or from containing 2,4-D, NA A and kinetin. The frequency of callus ini groups of ce ll s. In Lachenalia. the shoots were initiated mainly tiation was increased by the addition of yeast extract. This callus from single epidermal cells primarily derivatives of the stomatal produced small adventitious shoots and roots when transferred to mother ce lls. although a few shoots were ini tiated from groups of medium containing low concentrati ons of auxins and vitam ins cell s ( Niedcrwieser & Van Staden 1990b ). The frequency of (J ha & Sen 1984). mutations in shoots in itiated from single cells is usually fairly Although vast quantities of adventitious shoots can be pro high. resulti ng in ·solid mutations' (Hussey 1980). The adventi duced from callus, the frequency of genetically-aberrant shoots may be rel atively high. Thus, propagation via indirect adventi tious shoots also have been restricted to certain surfaces of tious shoots is usually avoid ed. In certain breeding programmes, cx plants. In IJ111riea. the shoots were initiated on the adaxial su r however, genetically-aberrant plantlets could be a useful source faces of bul b-scales. Thi s may be associated with anatomical dif of somaclonal variation. ferences between these surfaces. which included thicker cuticles, more chloropl asts and stomata on the abaxial surfaces, as well as Direct and indirect somatic embryos the presence of unidenti fi ed globu lar bodi es on the adaxial In Scilla, embryogenic callus was initiated on mediu m contain surfaces (Van Stadcn et a/ 1991). ing NAA and coconut mil k. The embryogen ic callus, which was Some adventitious shoots have been initiated on med ium con comprised of small thin-walled cells with large nuclei, formed taining no plant growth regulators, although most adventitious shoots and roots when transferred to medium contai ning no plant shoots have been initiated on medium containing various cytoki growth regulators (Chakravarty & Sen 1989). In Urginea. nins and auxins. In Ladtr:nalia. the add ition of BA had no influ embryogenic and non-embryogenic callus was in itiated on ence on shoot initiation in hybrids with high cytokinin-like medium containing 2,4-D and coconut milk. The embryogenic activity. but increased shoot initiation in hybrids with low cyto callus, wh ich was comprised of large, vacuolated parenchyma I.. inin-like activity (N iederwieser & Van Staden 1992). Further cells, formed greenish zones of small cells with large nuclei more, the substitution of cytokin in glucosides ([3G)B A, (7G)BA when exposed to 2,4- D for prolonged periods. These greenish & (9G] I3 A l for BA inhibited shoot initiation (Van Staden & zones produced globul ar embryos, which elongated to form Drewes 1994 ). In f/yacinthus, the addition of cytokinins had no banana-shaped bipolar embryos when transferred to medium influence on shoot initiation. although th e addition of auxins, containing low concentrations of BA with or without coconut particularl:-- IAA and IBA. increased shoot in itiation (Pi erik & milk. The bipolar embryos produced bulbous plantlets when Steegmans 1975: Bach & Cecot 1988, 1989). In Scilla, the add i transferred to medium containi ng kinetin and NAA. In Urginea, tion ofNA .~ inhibited shoot initiation (McCartan & Van Staden the age of the callus, the frequency of sub-culturing and the cyto 1998). while in Hyacintlms. the addition ofNAA promoted cal logical state ofthe callus influenced the initiati on and subsequent lus formation (Pierik & Steegmans 1975). In Hyacinlhus, the car development of the callus (Jha & Sen 1986). The ploidy of the bohydrate source also influenced shoot initiation with glucose callus (diploid & tetrap loid) also influenced med ium require and sucrose producing more shoots than fructose (Bach et a/. ments (Jha eta/. 1991). Despite the potenti al of thi s techn ique, 1992). In .lluscari, the add iti on of charcoal promoted bu lblet for somatic embryos have not been induced in many members of the mation and inhibited callus for mation (Peck & Cumming 1986), Hyacinthaceae. while i n/ ~\'(ld/1/h us . the addition ofpaclobutrazol promoted bul blet formation (Bach el a/ 1992). Some adventitious shoots pro Conclusions duced roots and/or bul bs spontaneously. Other adventitious The Hyacinthaceae comprises several genera, which are wide ly shoots have been rooted on med ium containing no plant growth exploited for their medicinal, pharmaceutical and ornamental regulators (Cook et a/ 1988). or various auxins, usually IBA potential. In South Africa, several members of the Hyacinthaceae (Nel 198 1. 1983: Taylor & Van Staden 1997) or NAA (Drewes are harvested without permits from wi ld popu lations, processed eta/ 1993: Drewes & Van Staden 1993). Although adventitious and then sold as traditional med icine. This is reducing the den shoots arc genetically less stable than ax illary shoots, particu sity, distribution and ge netic diversity of wild populations. Fur larly when they originate from si ngle cells, (Hussey 1980) this thermore, the enforcement of ex isting legislation has proved technique is frequently used to clone medicinal and ornamental ineffective with vast quantities of plants being traded locally and plants rapidly and economi cally. internati onally. Consequently, it has been suggested that ex situ S. A li·. J. Bot. 1999, 65(5 & 6) t.:onse::rvation through cultivation may alleviate pressures on natu Cape Province. pp. 8- 15. Books of Afri ca. Cape Town. ral resources, whilst meeting the demand for these plants. Con 13 LOM-BARNHOORN. G.J., VAN AARTRI.IK . .1 . & VAN DER ventional vegetative propagation, however, is usually fairly LINDE. P.C.G. 1986. Effect ofviraznle on thc production ofh)acinth slow. Micropropagation, therefore, provides a rapid and econom plants free li-mn hyacinth mosaic virus (I·IM V) by meristcm cultun;. ical means to propagate these endangered plants, whilst supply Acta Hort. 177:571-574. ing an alternative source of superior quality plants for the BRYAN, J.E. 1989. Bulbs. Vol. 2. pp. 324. Timber Press inc .. !long consumer market. Kong. Many bufadienolides have been isolated from members of the BUD/\ VARI. S. (Ed) 1996. The Merck. Index - an Encyclopedia of Hyacinthace::ae. These bufadienolides are used as cardi otonics Chemicals, Drugs and Biochemicals. 12th Ed. pp. 1352. Whitehou~c and, therefore. have pharmaceutical potential. This has led to the Station. NJ. CHAKRAVARTY. B & SEN. S. 1989. Regeneration through somatic identification and quantification of bufadienolides in various embryogenesis !rom anther explants of Scilla indica (Roxb.) 13akcr. clones and cytotypes in vitro and in situ to select high yielding Plant Cell Tiss. Org. Cult. 19: 71-75. strains suitable for commercial exploitation. In Bowiea, the same COOK. E.L.. CUNNINGHAM, A. & VAN STADEN . .1 . 1988. The us bufadienolides were accumulated at si milar concentrations for sue culture of an exploited Zulu medicinal plant, Bmviea mlubi!ts S both in 1·irro and in situ plants (Finnie et a/. 1994 ). In Urginea, Afr. J. Rot. 54: 509-5 10. however. the accumulation of bufadienolides was linked to the CRABTREE. D.G. 1947. Red Squill. thc most spcc ilic of the raticiucs. formation of specific organs in vitro, but was independent of the Eco. Bot. I: 394-40 I. genotype. explant source and regeneration system (Jha et a/. CUNN INGHAM. A. B. 1988. An invcstigiltion of the herbal mcdicinc I 99 I). Micropropagation, therefore, not only provides a rapid trade in Natal/Kwazulu. INR report 29. l' ictermantzhurg. and economi cal means to propagate selected chemotypes, but DAHLGREN, R.M.T., CLIFFORD. H.T. & YEO. P.F. 1985. Thc limu a lso a means to produce these bufadienolides in 1•itro. This is lies of the Monocotyledons- Structure, Evolution and Taxonomy. pp. important economically since it obviates the need to cultivate 129-133, 188- 193. Springcr-Vcrlag. Berl in. these plants ex virrum. DE HERTOGH, A.A. & I.E NARD. M. 199h World production ami Several members of the Hyacinthaceae are cultivated as orna horticnltural utilization of fl ower bulbs. In: Thc Physiology of f- lower rn e:: ntals. although the total area allotted to the production of llulbs, eds. A.A. De I lcrtogh and M. Lc Nard. pp. 21- 28. Elsevh:r. these bulbs is relatively small. Breeding programmes, which The Netherlands. have been established for certain genera, may increase the popu DE HER TOG H. A.A. & LENARD. M. 1993h. Gcnc:ral chapter on sum larity of these plants. Micropropagation, therefore, not on ly pro mer !lowering bulbs, In: The Physiology of Flowe::r Bulbs, cds. A.A. vides a rapid and economical means to propagate De Hertogh. and M. LeNard. pp. 74 1-774. Elsevier. The Ncthcrbnds. newl:r-devcloped cultivars, but also a means to eliminate viruses DREWES. F.E., BAYLEY. A.D. & VAN STADEN. J. 1993. Tissue cul sut.:h as Hyacinthus Mosaic Virus (B lom-Barnhoorn 1986) and ture of Schi:obnsis 111tricaw. a medicinal plant. S. Afr. J /Jot 59: I05 - Ornithogalum Mosaic Virus (Vcelar eta/. 1992), thus reducing 106. crop losses and faci litating the export of these plants. Microprop DREWES, F.E. & VAN STADEN . .1. 1993. In l'itro propagation of Ual tonia species. S. Afr J. Bot. 59: 457-458. agation also provides a means to conserve valuable germplasm DU PLESSIS. N. & DUNCAN, G. 1989. Bul bous plants of South ..:m (Louw 1995), therefore retaining useful character traits for future Africa. A Guide to their Cult ivation and Propagation. pp 71- 84 breeding programmes. Tatl!lbcrg. Cape Town. EL GRARI. R.. & 13ACKHAUS. R.A. 1987. In riu·o propagation of red Acknowledgements squill. Urgineonwntima Baker. Plant Cell Tis.1·. Org. Cult. I 0: 65-71. The:: National Research Foundation and the University of Natal FINN IE. .J .f.. DREWES. F. F.. & VAN STADEN . .J . 1994. 80\rnmmlu Re:: search Fund are thanked fo r financial assistance. Ms J.L.S. hi/is Harv. ex Hook.!: (Sea Onion): ht vitro cultun: uno prudu~:t i on of Taylor kindly provided Figures I A, F, G and H. cardiac glycosides. In: Biotechnology in Agriculture :md foorestr). Vol 28. Medicinal and Aromatic Plants VI I . cd . Y. l'.S. 13aja,i. pp References 84-97. Springer-Verlag. 13erlin . ARNOLD. T.H. & DE WET. B.C. 1993. Plants of Southem Africa: GENTRY. 1-l .S .. VERBISCA R. A. .l . & 13ANIGAN. T.F. 19!17. Red Names and Distribution, pp. 135, 146-155. NB I. Pretoria. Squill ( Urginea maritima, Liliaccue). Em. Bot. 41: 267-282. A Ul.T . .I .R 1995a. In vitro rooting and grccnhous~ ncclimntization of GOSLING. M. 1998. Muti mcccn taking root. Cape Times April 17. pp. l.adllmalia shoots. HortScience 30: 1304- 1305. 18. i\ lll.T.. l.l t 1995b. In vitro propagation of Eucomis aiiii/IIIIW!is. E IIANNWEG. K.. WATT. M.P. & 13ERJAK. P. 1996. A simple method t'omosa. and £. =ambesiocn by twin-scaling. HortSctence 30: 144 1- for the micropropngation of IJ01 riea \'Ofubifts li-\1111 intloreSCI! nCe 1442. explants. Bot. Bull. Aend Sin. 3 7: 213-218. i\ ULT ..l.lt 1996. In vitro rooting nnd greenhouse acclimatization of HUSSEY. G. 1975. Propngation nf hyilcinths by tissue culture. S". I 'elrheunia hracteata and V. capensis shoots. HortScience 31: 1229- Hart. 3: 21-28. 1230. HUSSEY. G. 1976a. ln1•ifro rele JAGER. A.K. & VAN STADEN. J. 1995. Screening lor cardiac glyco· & Org. Cult. 22: 223-228. sides in Schb1husis il1lricata. S. Afr. J. Bot. 61: I 01 - 103. NIEDER WIESER. J.G. & VAN STADEN . .1 . l990h. Origin nfmhcnti· .lilA. S. 1988. 13ufadiennlidcs.ln: Cdl Culture and Somatic Cell Genet· tious buds on cultured l.achenalm leaves. Be11r Bwl. 1'/lcm:en 65: ics of Plants. Vnl 5. Phytocht:micals in plant cell cutures. cd. l.K. 443-453 . Vasil. pp. 179-191. Academic Press Inc .. Flu. NIEDER WIESER . .I.Ci . & VAN STADEN.. 1. 1992.1nteraction hetwccn .IliA. S .. MITRA. G.C.. & SEN. S. 1984./n vuro regeneration from bulb bcnzyladenine. naphthalcneacctic acid and tissue age on ad\·entil ious explunts of Indian squi ll. Urgmea indica Kunth. Plant Cell Tiss. Org. bud formation on leaf sections of l.achenalia hybrids. S Aji· J /Jot Cult. 3:91- 100. JfiA. S .. SA II U. N. P. . & MAHATO. S.B. 1991. Callus induction, orga· 58: 13-16. nngencsis and somatic embryogenesis in three chromosomal races of NIEDERWIESER. J.G. & VCTI.AR. B.l\1. 1990. Regeneration of llrginea nullca and production of hui~Idiennlides. Plam Cell Tiss. Lachenalia species I rom leaf ex plants. llorJ51m:nce 25: 684-6!\7. Org. Cult 25: 85-90. PECK. D.E. & GUMMING. 13.Ci. I986. Bcnetkial effects of act inned JHA. S. & SEN. S. 1981. Bufadienolides in different chromosomal races charcoal on bulblet production in tissue cul tures of Muscan armenia· of Indian Squill. Phytochemistrv 20: 524-526. cum. Plant Cell ?iss. Org. Cult. 6: \J- 14 . .IHA. S. & SEN, S. 1983. Quantitation of principal bufadicnolides in dit~ PERRY. P. 1985. The restmcturing of the t~unily l.iliaccac. f"e/d & lercnt cytntypes of Urginea indica. Planta Med1ca 47: 43-45. Flora 7 1: 66-68 . .IliA. S & SEN. S. 1985. Regeneration and rapid multiplication of Boll'· !'!ERIK, R.L.M .. & POST. A.J.M . 1975. Rapid vegetative propagation 1ea voluhdis Harv. in tissue culture. Plant Cell Rep. 4: 12-14. of f~vacmthus orienta/is L. in varo. Sci. firm. 3: 293-2'.17 . .lilA. S. & SEN. S. 1986. Development oflndian Squill (Urginea mdica !'I ERIK, R.L.M. & STEEGtv1ANS. H.H.M. 1975. Etlecl ofau:-.ins. cyto Kunth .) through somatic embi)"ogencsis from long tcm1 culture. J. P/a/11. Physio/. 124: 43 1-439. kinins, gibbcn:llins. ubscisic acid and ethephon on regeneration and JON ES. N.IJ .. MITCHELL . .1., BAYLEY. A.D. & VAN STADEN. J. growth of bulb lets on excised bulb scale segments of Hyacinth. /'hys I992. ln1·itm propagation of l'llllmntlws baswicum. S. Afr. J. Bot. 58: iol. Plant. 34: 14-17. 403-404. REES. A.R. 1992. Onmncntul bulbs. corms and tubers. pp. 93- 11 I. l.ANDDY. P.A. & N!EDERWIESER. J.G. 1992./nvitro propagation of CAB lnternutionul, UK. Omitlwgalum 'Rollow'. J. S Afr. Soc. Hort. SCJ. 2: 50-54. Sl!YR. S.E. & STAB A, E.J. 1976. Examination of Squi ll tissue cult ures I.E NARD. M. & DE HERTOGH. A.A. 1993. General Chapter on ll.1r bufadicnolidcs and anthocyanins. /'Iaiita Mec!Jca 29: 86-90. Spring-flowering Bul bs. In: The Physiology nf Flower Bulbs. cds. TAYLOR. J.L.S. & VAN STADEN . .1 . 1997. In wlro propagation of A.A. De llt!rtogh and M. Lc Nard. pp 705-739. Elsevit:r, The Nether· Ve/Jheimia hracteata and V bracteara 'l.t:mon Flame'. S. .·!fr . .I fim hinds. 63: 159-161. l.OUW. E. 1995. Long tcm1 storage of Lachena/ia shoot tips. J. S. Afr TAYLOR. J.L.S. & VAN STADEN. J. 1998. Micropwpagation of Soc. Horl. Sci. 5: 77-80. l.UYT. R.P .. JAG ER. A.K. & VAN STADEN. J. 1999 The rational Eummis species: a plant with horticultural and medicinal pot.:nti al. IX usage of /Jrimia robusta Dak. in traditional medicine. S. Afr. J. Rot. 111/emationa/ Congress on Plalll Tissue & Cell C'uflllre . .l~ r usalcm. (In Press). Israel. MANDER. M.. MANDER . .1 .. CROUC H, N.. McKEAN. S. & VAN HORN. D.l.. & DOMINGO. W.E. 1950. Comparison of seed