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 = benzyladenine, 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 The Asparngales forms a large and fairly homogenous complex of fami lies. It is believed to be a monophy letic group, which evolved in parallel with the Li liales and Dioscoralcs. The Aspar­agales consists of 31 fami l ies including several new families. These fami lies. which include the All iaceae, Asparagaceae, Asphodelacene, Dracennaceae, Eriospermaceae and Hya­cinthaceae. compri se genera previously placed wi thin the Lil­iaceae Wll.\'11 fato (Dahlgren et a/. 1985; Perry 1985). The Hyacinthaccae comprises several genera including Bo111iea. F.u~·omis, Lachenalia, Ledebouria, Scilla and l/rg inea. These genera arc charncterised by bulbs with thick and sometimes con­tractile roots. basal leaves and simple or more rarely branched racemes. The inflorescences comprise actinomorphic, bisexual tlowers, which range from white, blue, violet, and more rarely yellow, red, brown to nearly black. T he Hyac inthaceae are w idely distributed, occurring in southern Africa and in a region from tht: Mediterranean to SW As ia (Dahlgren et af. 1985; Duncan & du Pless is 1989).

Medicinal and pharmaceutical potential

In South A fr ica, approximately 20 000 tons of plant material , worth about R270 million, is harvested, processed and sold as traditional medicine annually (Gosling 1998}. Approximately 14% of this plant material comprises bulbs (Mander 1997}, which are destruct ive ly harvested, processed and sold for the treatment of var ious ai lments (Table I ). These bulbs, which are sold whole, sliced or chopped, are usually administered as decoc­tions, emetics and enemas (Watt & Breyer-Brandwij k 1962; Jacot Gui llamod 1971; Hutchings 1989; Mander et af. 1995; van

Wyk eta/ 1997). $'cilia natalensis is one of the top-ten most popular medicinal

plants in South Africa (Cunningham 1988; Williams 1996; Man­der 1997). Despite its specially protected conservation status. approximately 95 tons of these bulbs are sold illegally in Durban annually. The price of these bulbs is relatively low ranging from R 1.89 to R6.80 per kilogram. This is mainly due to the occur­rence of large populations of these bulbs locally. T he price of another popular medicinal plant, Bowiea volubilis is substan­tially higher ranging from R 11.74 to R27.80 per kilogram (Mander 1997). The price of these bulbs has increased steadily wi th the decline in their avail abi lity and size (Cunningham 1988).

A lthough the active ingredients of many of these bulbs have not yet been identified. several bufadienol ides have been iso lated from members of the Hyacinthaceae including Bml'iea I'Oiubilis (Jha 1988; Finnie et a/. 1994), Drimia rohu.1·ta (van Wyk eta/ 1997; Luyt et a/. 1999}, Urginea a/tissima (Hutchings et a/. 1996), U. indica (Jha & Sen 198 1, 1983; Jha 1988) and (} mar­itima (Shyr & Staba 1976; Verbiscar eta/. 1986; Gentry el a/ 1987; Jha 1988). Bufadienol ides such as proscillaridin A are val­uable since they can be administered to digital in sensitive/intol­erant patients (Jha 1988). These bufadienol idcs. however. are usually not used as cardiotonics due to their low therapeutic indi­ces (Jager & van Staden 1995). However, some bufadienolides identified in certain organs of Bowiea l'oluhi/is are thirty to sixty ti mes more active than those fi·om Digitalis (Hutchings et a/ 1996). Proscillaridin A, which is derived by enzyme hydrolys is from sci llaren A , is produced under several trade names includ­ing Caradin®, Cardion®, Proscillan®, Sandoscill®, Scillac­rist®, 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 namc1

Alhuw mnadensis (L) Leighton syn. A. JllllfOI"

Medicinal uses

As an anthelmintic, thirst quencher anc.l to treat venereal c.liseases

References

Watt & Brcyer-Brandwijk I 962; Hutchings 1989

Albuca coopen Bak. As an anthelmintic, lotion for washing wounds and Watt & Breyer-Brandwijk 1962; llutchings to treat venereal diseases 1989

To treat illnesses caused by poisons Hutchings eta/ 1996 .·1/huca fusugaw ( L.f.) Dryanc.l.

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

Bml"iea m/uhilis Harv.

/Jtpcatli hreviofo/ium (Thunb.) Fnurc.

As an anthelmintic and to treat constipation and gonorrhoea

To treat c.lropsy. barrenness in women and head­aches

To treat heart pains and breathlessness

fJtpcatlt gmcillimum Bak. syn. /J. po~vphyf- To treat gonorrhoea and pimples fum

Oipcaclt \'ll"tde (L.) Moench

!Jrimia ctliaris Jacq.

f)rimia data Jacq.

/)nmiallem!formi.l· Bak.

f)rimia mhusta Oak. syn. D. alw

Ez""OIII/.1"1111111111/Utlis (Mill.) Chill.

f:"ummts hicolor Bak.

To treat gonorrhoea and flatulence

As an emetic. expectorant and diuretic

To treat high blond pressure and stabbing pains

To treat extemal tumours that have been lanced

As an expectorant, emetic and enema to treat feverish colds

To treat co lic. llatulence, syphilis and hangovers

For colic and as a purgative

f:'ucomts mmo.w (Houtl.) var. comosa syn E. Tn treat rheumatism and teething infants JIIIIIC/(1/(1

t:ucomts JIO/eevansii N .E. Br.

f:"ucoll/1.1" regia (L.) L 'Heril

f.l!clehrwria cooperi (I look. I:) Jessop syn. Scilla cooperi. S. immdensis. S. satum

f.edehmll·w ovatifolia (Oak.) Jessop syn. Scilla m·wifolia

l.edehouno rewJ!wa (I.. f.) Jessop syn. Scilla

faiiCI!III!(Ofill

.\/a.uonut echinata L.f. syn M . bowker1

Ormtlwgalum longl/Jractealllm Jacq.

For people sut1'ering from mental disease

To treat venereal disease, lumbago, diarrhoea, respiratory conditions especially cough nnd biliousness and to prevent premature childbirth

Used in the initiation ceremony of boys; to treat gastro-intestinal, gynaecological and psychologi­cal ailments

To treat gastro-intestinal and gynaecological ailments, backache and inllucnza

For treating lumbago and gall sickness in animals; to bathe skin eruptions anti as an ointment for wounds and sores

ror ophthalmic applications. sterility and tooth­ache

To treat swellings or growths

Ormtlwgalum duhiwn Houtt. syn 0. mima- As an anthelmintic ll/111

Ormtlwgalum lr!lllljolium De Iaroche subsp. As a rat poison tenui(ofiunz syn. 0. eckfonii & 0. viren.1·

Ormthoj<alum thyrsoides .J acq. To treat diabetes mellitus

Schiznbasis intricala To treat chest complaints

Walt & Breycr-Brandwijk 1962; Jacot Guil­lamnd 1971; Hutchings 1989

Walt & 13rcycr-l3rantlwijk 1962; Batten & Bokclmann 1966; Hutchings 1989; Hutch­ings eta/ 1996

11utchings 1989; Hutchings et al 1996

Watt & 13rcycr-13randwijk 1962

Watt & Breycr-Brandwijk 1962; l3attcn & Bokelmann 1966: Hutchings 1989; llutch­ings et a/ 1 996

Walt & Breyer-Brandwijk 1962

Hutchings el af 1996

Watt & Breyer-Brandwijk 1962; Jacot Guil­lamod 1971

Hutchings 1989; Hutchings et af 1996

Hutchings et af 1996

Watt & Breyer-Brandwijk 1962; Batten & 13okclmann 1966; Hutchings et af 1996

Walt & Brcyer-Brandwijk 1962; Batten & l3okclmann 1 966; llutchings 1989: Hutch­ings eta/ 1996

Wall & Brcycr-Brandwijk 1962

Watt & l3rcycr-Brandwijk 1962

Watt & Brcyer-Brandwijk 1962; Hallen & !3okclmann I 966: Hutchings 1989: Hutch­ings et a/ 1996

llutchings 1989; Hutchings et of 1996

Watt & Brcyer-Brandwijk 1962: Jacot Guil­lamod 197 1

Walt & Breyer-Brandwijk 1962; Jacot Guil­h:mod 1971

Hutch ings 1989: Hutchings et a/ 1996

BaHcn & Bokelmann 1966

Watt & Brcyer-Brandwijk 1962: Jacot Guil­lamod 197 1

Watt & l3reyer-13randwijk 1962

Hutchings et of 1996

Scillallatafellsis Planch. syn. S. kraussii & S. As a purgative anti to treat sprains, fractures. boils. Hutchings cl a/ 1996 dracomo11tana vcldsorcs and lung sickness in cattle

Set! fa nermsa (Burch. ) Jessop syn. S. rigidi- To treat fevers, gastro-intestinal ailments and folia insanity

Bryant 1966; Hutchings I 989; Hutchings et a/ 1996

S. Ati". J. Bot. 1999, 65(5 & 6) 363

Table 1 Continued

/ .'rgineaaltJssima (I.. f.) Bak. To treat rheumatic swellings, gastro-intestinal and Watt & 13rcycr-Brandwijk 1962; Hutchings 1989; Hutchings eta/ 1996 respiratory ailments

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 delivery during birth

Hutchings I 989: llutchings eta/ 1996

l ."rgmea rubella Bak. For the treatment of colic Watt & Brc)cr-Brandwijk 1962: Jacot Cluil­lamotll971

l r~;mea .wmgumea Schinz. syn. li. burkei As an abortifacient and lo treat paralysis, circulatory diseases and rheumatism pains

Watt & 13rcycr-Brantlwijk 1962

' Plant name and synonyms (Amok! & De Wet 1993).

glycosides increase the force of the systolic contraction, inhibit lhe atrioventricular conduction, and enhance the 'automacy' of the cardiac tissue and. therefore, are used to treat heart fai lure and certain arrhythmias (Walton eta/. 1994).

13ufadienolides such as sci ll iroside and scillaren A also are effective rodenticides with the French Pharmacopoeia Codex describing a raticidal paste composed of powdered bulb of Red Squill ( LlrKinea maritima), fl our and sugar (Balbaa eta/. 1979). The erticicncy of these strongly emetic rodenticides is largely due to the inability of rodents to regurgitate. This renders these products extremely safe and specific since humans and domestic animals including chickens, cats and dogs readily regurgitate accidentally-i ngested baits (Crabtree 1947). In the 1940's, sev­eral tons of powdered bulb of Red Squill ( Urginea maritima) were imported into the USA. This ceased with the introduction of warfarin and other anticoagulant-type rodenticides. The devel­opment of warfarin-resistance in rodents, however, has led to renewed interest in Red Squill-formulated products as co-roden­ticides ( V crbiscar eta/. 1986). This also has led to the identifica­tion and quantification of bufadienolides in various clones or cytotypes of Indian Squill (U indica) (Jha & Sen 198 1, 1983) and Red Squill ( li. maritima) (Gentry et a/ 1987) to select high-yielding strains suitable for commercial exploitation.

Ornamental potential Approximately 16 000 ha are allotted to ornamental bulb produc­tion in the Netherlands, representing 55% of the world 's total production area, with significantly smaller areas allotted to orna­mental bulb production in the USA (4 449 ha), the UK (4 300 ha). Japan ( I 622 ha), France ( 1 285 ha) and South Africa (425 hnl (De Hertogh & Le Nard 1993a). Despite the ornamental potential of the Hyacinthaceae. the area allotted to the production of /~un1111is (2 ha), Galtonia (I ha), 1/yacint/ws (955 ha), Orni­tlwga/um (50 ha), Scilla (20 ha) and Urginea (3.2 ha) is small (De He11ogh & LeNard 1993 a, b; Le Nard & De Hertogh 1993 ), representing approximately 3% of the world 's total ornamental bulb production area. Many of these genera including Hyacin­thus. Lachenalia and Scilla are worthwhile pot plants, while oth­ers including Eucomis. Ornitlwgalum and Urginea are useful garden plants (De Hertogh & Le Nard 1993b; Le Nard & De Hertogh 1993 ). A breeding programme for Lachenalia compris­ing five phases including the establishment of a genebank, and the development and subsequent evaluation of hybrids for com­mercialization has been established over the past thirty years (Nicderwieser eta/. 1998 ). The successful introduction of these hybrids onto the international flower market could initiate other breeding programmes, increasing the popularity of these and other plants.

Conventional propagation Several members of the Hyacinthaceae, therefore, have potential as alternative horticultural/agricultural crops, providing crops of both bulbs and inflorescences. Seed, however, is seldom used for commercial propagation, except where the species or cultivars produce large quantities of reliable seed with short juvenile peri­ods (Rees 1992). Some members of the Hyacinthaceae including Scilla siberica, S. siberica cv • Alba', and S. bifolia, however, are only propagated by seed, while others including S. siberica cv 'Spring Beauty' and S. tubergeniana [= S mischtschenkoana (Bryan 1989)] are propagated vegetatively (Le Nard & De Her­tagh 1993). Although offsets are used to propagate many mem­bers of the Hyacinthaceae, this is generally too slow for commercial propagation. Several artificial techniques, which include scaling, basal cuttage (scooping and scoring) and bulb cuttings, are used to increase the rate of natural multiplication. These, however, are usual ly restricted by the size of the mother bulb.

Micropropagation Several members of the Hyacinthaceae have been micropropa­gated using various techniques. These include the proliferation of axillary shoots, the ini tiation of adventitious shoots and the induction of somatic embryos, which are discussed in greater detail (Table 2). Some details, however, such as the frequency of shoot initiation, the average number of shoots initiated, the fre­quency of fasciated, deformed or mutated shoots, the frequency of plantlets successfully acclimatized, and the propagation potential within a specified time-frame are seldom reported. These details, which should be included routinely in micropropa­gation reports, are essential for commercial fac ilities.

Axillary shoots In Eucomis. axi llary shoots were proliferated from twin-scale explants, which were cultured on medium containing combina­tions of BA and NAA. These shoots, which were then sub-cul­tured onto the same medium, proliferated more shoots resulting in a continuous culture system. After 8- 10 weeks, the shoots were then rooted on medium with or without NAA (Ault 1995b).

Although axillary shoots are genetically-stable, the limited availability of these axillary shoots restricts the potential of this technique (Hussey 1980). Furthermore, 'mixed' cultures com­prised of axillary and adventitious shoots do occur occasionally (Hussey 1976a). The origin of these axillary shoots is seldom confirmed, and thus, the frequency of 'mixed cultures' is rarely reported.

Direct adventitious shoots In several members of the Hyacinthaceae, adventitious shoots 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

/Jowiea \'oluhilis Bulb sc<Jlcs Shoots

/Jourea m/uhrlts In llon:sccnce Buds and callus

/Jou iea mluhilis lntlorescence

Bulb-scales ~nd split shoots

Shoots Shoots

Bowrca l'olubilts Twin-scales

Orimia rollltsta 13ulb-scalcs Rulblets

Medium an<.! supplcments 1

MS. Sucrose (30000), 13A ( I). NAA (I), Agar (8000} MS. Sucrose (30000}. Agar (8000}

MS. Sucrose (30000}. HA (I). 2.4-D ( I). Agar (I 0000) MS. Sm:rose (30000). Agar ( 10000)

Growth response

Shoots Plantlels

Shouts Bul b lets

Mo<.litic<.l MS. Sucrose (30000}. CM ( 150). 2.4-D (I ). Ag<~r Callus (6000) Modi lied MS. Sucrose (30000). Cll ( 100). 2.4 -D (0.5), Shoots Agar (6000) Modi tied MS. Sucrose (30000), Agar (6000) l'lantlets Modi fied MS. Sucrose (30000). BA (2), 2.4-D (0.05) l'lantlets

MS. Sucrnsc (30000). BA ( I 0). NAA ( I), Agar (8000)

MS. Sucrose (30000}, 13A (2), NAA ( 1 ). Ag<~r ( 1 0000) MS. Sucrose (30000). NAA ( 1 ). Agar ( 1 0000)

Shoots

Bulblets Root..:d bulblcts

J:ucomi.~ att1wn1w- Twin-scales H·om bulbs MS. Sucrose (30000), BA (0-5). NAA ( 1 ), Agar (7000) Shoots 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), Gel rite (2000) Shoots h I:· mnw.1·a. E. MS. Sucrose (20000). lA A ( 1-2) or I BA ( 1-2), Gelrite lun>lor, E lmmilis. Shoots (2000) l'hmtlets E. =ambeswca (rigure lA)

Refi:rences

Cook t'/ a/ I 988

llannweg et a/ 1<)96

.lim & Sen !9!15

Van Staden e1 a/. (991

Ngugi era/ I <Jl)8

Aull 1995b

Ta)lor& Van Stndcn 1998

Eun mrt.1· polccmn- Seeds Modified MS, Sucrose (20000), Gdritc (2000) Secdl in~:s McCartan & Van stt Leaves of seedli ngs

Shoots Mndi lied MS. Sucrose (20000), NAA ( 1), Gelrite (2000) Modified MS. Sucrose (20000). lA A ( 1 ), Gel rite (2000)

Shoots (Figure 1 Bb) Staden I 995 Planllcts

Eucomt.l \'(}}lder-1111!1' \1'1..'1

( ialtonio <'llllllt­

mlls & (i l'trldr­j/om

Leaves Shoots

l'e<.liccls & ovary bas~s Split shoots

lf.racttllhus orren- Leaves ta/1.1· cv. Delft's 131u~

I f.mcmtlm.t cmen- Basal parts nf bulbs talis cv. Pink Pearl

Modi fied MS. Sucrose (20000). 13A (1-2), lAA (I). Geln te Shoots (2000) Pluntlcts Modi tied MS. Sucrose (20000). 113A (I), Gdrite (2000)

MS. Sucrose (30000). BA (0.3),NAA ( 1 ), Agar (8000) MS (Y: ). Sucrose (30000). NAA (0.5). Agar (8000)

Shoots Plantlcts

MS. Glucose or Sucrose (30000). 13A ( l ). NAA (0. 1 ). ± 13ulblcts PAC (8), Ag<~r (8000)

Modi lied Knops (V.,). Glucose (20000). lAA ( 10). Agar 13ulblcts (6000)

flyad mhu.1· sp cv. Twin-scales ti·om bulbs MS. Sucmsc (20000). Agar (7000) Shoots 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

l.uchenolw Leaves arhurllllollll<'. I. Shoots l>ulhifem . I. pur­fJIIrt'c>c·oemlca

l.adrenu!ta cv. Rohyn. Rolina. Romargo & Romaud

l.acherwlw cv. Rnmaud

l.adlellalw hybrids

Lachellulw hybrids

Leaves

Leaves

Leaves

Leaves Shoots

l.achenalw spp. & Le<Jves hybrids

MS. Sucrose (20000). Agar (7000)

MS. Sucrose (30000). 13A (2). K-NAA (0.1 ). Ag<~r (6000) Shoots MS. Sucrose (30000). K-IBA (0-2) or K-NAA (0-2). Agar Plantlcts (6000)

Modi tied MS. Sucrose (I 0000-90000). BA (2). NAA (0. 1 ). 11u<.ls Agar (7000)

Modified MS. Sucrose (50000). BA (4). NAA (2). Agar Buds (7000)

MS. Sucrose (30000). BA (2). NAA (0.1 ). Agar (6000) 13uds

Modi lied MS. Sucrose (30000). 13A (2). NAA (0.1 ). Agar Shm1ts Phmth::ls (7000) Modi tied MS. Sucrose (300fl0). lBA (2), Agar (7000)

Mo<.litic<.l MS. Sucrost:: (50000). RA ( 1 ). NAA ( 1 ). Agar Buds (6000)

McCartan et of. (In Press)

Drewc:s & V;m Staden 1993

Bach et a/. I <)')2

Pi erik & Stcc:g­mans 1975

Hussc} 1975

Auh 19<J5a

Van Rcnshurg &. Vcelar l 98~

Van Sta<.len & Drewes 199<1

Nic<.lcm ciser & Van Stadcn 1992

Nd 1983

N J<.:derweiser & Van Stac..lt:n 1990h

S. Afr. J. Bot. 1999, 65(5 & 6)

Table 2 Continued

lfmcari armenia- 13ulb scales cw11 cv. Early (jiant

Ormthogalum cv. Leaves Rollmv

Ornithor.alum cv. Leaves Rogel Meristems from buds

()mithogalum hybrid

On1ithoga/unl IIIOCII/lllllll/

( Jmithogalwn tlm·.wides

Omithogalum umhellatum

Sclli:ohmis ino·i­cctla

Leaves Shoots

Leaves

Stem, Leaves. Ovary, Sepals, Bulb scales

Twin-scales of bulbs Callus Shoots

Bulb Scales Shoots

Sed/a hyacinthi- Leaves ww Shoots

Shoots

Scilla nawlensi.~ l3ulbs ( Figun: I C) Leaf and bulb scales of

shoots Shoots

/lwmntho.f basuti- Bulb scales Cl/111

/ .'rgmea indica (2n. 3n 7 4n}

/ .'rgtnea mdica

Twin-scales of bulbs Callus Callus

Twin-scales of bulbs Somatic embryos Plantlets

Modi lied MS, Sucrose (30000). BA (5}, NAA (2}. AC ( I 000). Gel rite (2000}

Bulblets

Modified MS, Sucrose (30000}, BA (2}, NAA (0.1 }, Agar Shoots (7000}

Modi tied MS. Sucrose (30000}, BA (2}, NAA (0.1 ). Gel rite Buds (2000) Virus-indexed plant-Modilied MS, Sucrose (30000), BA (I}, NAA (I}, adenine lets arabinose ( I), Gelrite (2000}

Modi lied MS, Sucrose (30000), BA (2), NAA (0. 1 }, Agar Shoots (7000} Plantlcts Modified MS. Sucrose (30000), Agar (7000}

Modified MS, Sucrose (30000). BA (2), NAA (0.1). Agar Callus & shoots (7000}

MS. Sucrose (20000), Agar (7000)

MS, Sucrose (30000),NAA (8}, Ag;~r (5000) MS. Sucrose (30000), BA (0.5), NAA (2), Agar (5000} MS ('/>}, Sucrose (30000), Agar (5000)

MS, Sucrose (30000), BA (2). NAA (2), Agar (8000) MS (1/:), Sucrose (30000), NAA ( I), Agar (8000)

Plantlets

Callus Shoots Plantlcts

Shoots and callus Plantlets

365

Peck & Cumming 1986

Landby & Nicd­erweiser 1992

V celar et a/ I 992

Nel 198 1

Van Rcnsburg et a!. 1989

Hussey 1976h

Nayak & Sen 1995

Drewes eta/. 1993

MS, Sucrose (30000), Agar (I 0000) Shoots Nair 1989 MS. Sucrose (30000), Kinetin (5), NAA ( I), Agar ( 10000) Shoots MS, Sucrose (20000) Plantlcts

Modi tied MS. Sucrose (20000), Gelrite (2000) Shoots McCartan & Van Modi tied MS. Sucrose (20000), Kinetin ( 1-2), IAA ( 1-2), Shoots (figures I D Staden 1998 Gelrile (2000) & E) Moditicd MS, Sucrose (20000). IAA (I), Gel rite (2000) Plantlets

MS. Sucrose (30000), BA (0.1 ), NAA (5), Agar (8000) Ptantlcts Jones eta/. 1992

Modi tied MS, Sucrose (30000), Kinetin ( 1-2), 2,4-D ( 1-4), Callus depending on Jha et a/. t 991 ± NAA (2), ± CM ( 150), ± YE ( I 000), Agar (6000} ploidy 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

Moditkd MS. Sucrose (30000). 2,4-D (2), CM ( 150), Agar Callus and somotic Jha ct a/. 1986 (6000) embryos 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 Bulblets

Modified MS, Sucrose (40000), BA (0.1-0.3), NAA (0. 1- Plantlets 0.3 ), Agar (7500) Bulblets Modified MS, Sucrose (40000), NAA (0.1-0.3), Agar Ptantlets (7500}

El Grnri & Back­haus 1987

l"cltheimia hracte- Leaves and nora! stems MS. Sucrose (30000). BA (2), NAA (0.1 ), Agar (7000) Shoots Plnntlels

Ault 1996 Ilia cv. Lemon Shoots MS. Sucrose (30000), K-NAA (0-2), Agar (7000) 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 Plantlets (Figure I H) Staden 1997 ala (Figure t F) Shoots MS, Sucrose (30000), IBA (2), Agar (8000)

1 Figures in parentheses indicate mg 1·1 or m11·1•

explants. T he explant source, orientation, position and s ize, how­ever, influenced shoot initiation (Niederwieser & Van Staden t 990a & 1992: Niederwieser &Vcelar 1990; Landby & Niederw-

ieser 1992). In Scilla, the explant source influenced shoot in itia­

tion with leaf ex plants producing more shoots than bulb explants (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 adaxia l 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)

cxplants intluenced shoot initiation, with young ex plants produc­ing more shoots than old explants. This may have been linked to endogenous cytokini n levels particularly in the intermediate and old explants (Niedcrwieser & Van Staden 1990a). In Ornithoga­lum, the orientat ion of the explants also influenced shoot initia­tion with <~polar explants producing more shoots than polar ex plants ( Landby & Niederwieser 1992), whi le in Lachenalia. callus and deformed shoots were formed when explants were cultured with the adaxial surface down (Niederwieser & Vcelar 1990). In l.ucllenalia, the explant size influenced shoot initiation wi th small explants producing more shoots than large explants. The optimum size explant was 3.3 x 15 nun {Niederwieser & Vcelar 1990). This may have been related to the wound surface, which is proportionally larger in smaller explants. In Omithoga­lum. however. additional wounding on the surface of the explant inhibited shoot initiation (Landby & Niederwieser 1992). In I ~mcimlws. the explant size also influenced shoot initiation, which decreased linearly with a decrease in explant size (Pierik & Post 1975).

These adventitious shoots originated from single cells or from groups of ce lls. In Lachenalia. the shoots were initiated mainly from single epidermal cells primarily derivatives of the stomatal mother cells. although a few shoots were ini tiated from groups of cells ( Niedcrwieser & Van Staden 1990b ). The frequency of mutations in shoots initiated from single cells is usually fairly high. resulti ng in ·solid mutations' (Hussey 1980). The adventi­tious shoots also have been restricted to certain surfaces of cxplants. In IJ111riea. the shoots were initiated on the adaxial sur­faces of bulb-scales. This may be associated with anatomical dif­ferences between these surfaces. which included thicker cuticles, more chloroplasts and stomata on the abaxial surfaces, as well as the presence of unidenti fied globular bodies on the adaxial surfaces (Van Stadcn et a/ 199 1 ).

Some adventitious shoots have been initiated on medium con­taining no plant growth regulators, although most adventitious shoots have been initiated on medium contain ing various cytoki­nins and auxins. In Ladtr:nalia. the add ition of BA had no influ­ence on shoot initiation in hybrids with high cytokinin-like activity. but increased shoot initiation in hybrids with low cyto­I..inin-like activity (Niederwieser & Van Staden 1992). Further­more, the substitution of cytokinin glucosides ([3G)BA, (7G)BA & (9G] I3A l for BA inhibited shoot initiation (Van Staden & Drewes 1994 ). In f/yacinthus, the addition of cytokinins had no influence on shoot initiation. although the addition of auxins, particularl:-- IAA and IBA. increased shoot in itiation (Pierik & Steegmans 1975: Bach & Cecot 1988, 1989). In Scilla, the addi­tion ofNA .~ inhibited shoot initiation (McCartan & Van Staden 1998). while in Hyacintlms. the addition ofNAA promoted cal­lus formation (Pierik & Steegmans 1975). In Hyacinlhus, the car­bohydrate source also influenced shoot initiation with glucose and sucrose producing more shoots than fructose (Bach et a/. 1992). In .lluscari, the add ition of charcoal promoted bulblet for­mation and inhibited callus formation (Peck & Cumming 1986), while in/~\'(ld/1/hus. the addition ofpaclobutrazol promoted bul­blet formation (Bach el a/ 1992). Some adventitious shoots pro­duced roots and/or bulbs spontaneously. Other adventitious shoots have been rooted on medium containing no plant growth regulators (Cook et a/ 1988). or various auxins, usually IBA (Nel 198 1. 1983: Taylor & Van Staden 1997) or NAA (Drewes eta/ 1993: Drewes & Van Staden 1993). Although adventitious shoots arc genetically less stable than axillary shoots, particu­larly when they originate from single cells, (Hussey 1980) this technique is frequently used to clone medicinal and ornamental plants rapidly and economically.

367

Callus and indirect adventitious shoots In Boll'iea, callus was initiated on medium containing 2.4-D and coconut milk, and then transferred to medium containing 2,4-D and casein hydrolysate. This callus was comprised of green nod­ules, which produced adventitious shoots, when transferred to medium containing low concentrations of 2,4-D (Jha & Sen 1985). In Omithogalum. callus was initiated on medium contain­ing NAA alone (Hussey 1976b; Nayak & Sen 1995) or in combi­nation with BA (Van Rensburg et a!. 1989). The frequency and size of the callus decreased with increasing sucrose concentra­tion (Van Rensburg eta/. 1989). Hussey (1 976b) found that the callus was comprised of a mixture of diploid and tetraploid cells. He also found that the frequency of tetraploid cells increased with callus age (Hussey 1976b). In contrast, Nayak and Sen ( 1995) found that the callus was genetically-stable for several years. The callus produced adventitious shoots when transferred to medium containing no plant growth regulators (Hussey 1976b) or medium containing combinations of BA and NAA (Nayak & Sen 1995). In Urginea, callus was initiated on medium containing 2,4-D, NAA and kinetin. The frequency of callus ini­tiation was increased by the addition of yeast extract. This callus produced small adventitious shoots and roots when transferred to medium containing low concentrations of auxins and vitamins (Jha & Sen 1984).

Although vast quantities of adventitious shoots can be pro­duced from callus, the frequency of genetically-aberrant shoots may be relatively high. Thus, propagation via indirect adventi­tious shoots is usually avoided. In certain breeding programmes, however, genetically-aberrant plantlets could be a useful source of somaclonal variation .

Direct and indirect somatic embryos In Scilla, embryogenic callus was initiated on medium contain­ing NAA and coconut milk. The embryogenic callus, which was comprised of small thin-walled cells with large nuclei, formed shoots and roots when transferred to medium containing no plant growth regulators (Chakravarty & Sen 1989). In Urginea. embryogenic and non-embryogenic callus was initiated on medium containing 2,4-D and coconut milk. The embryogenic callus, which was comprised of large, vacuolated parenchyma cells, formed greenish zones of small cells with large nuclei when exposed to 2,4- D for prolonged periods. These greenish zones produced globular embryos, which elongated to form banana-shaped bipolar embryos when transferred to medium containing low concentrations of BA with or without coconut milk. The bipolar embryos produced bulbous plantlets when transferred to medium containing kinetin and NAA. In Urginea, the age of the callus, the frequency of sub-culturing and the cyto­logical state ofthe callus influenced the initiation and subsequent development of the callus (Jha & Sen 1986). The ploidy of the callus (diploid & tetraploid) also influenced medium require­ments (Jha eta/. 1991 ). Despi te the potential of this technique, somatic embryos have not been induced in many members of the Hyacinthaceae.

Conclusions The Hyacinthaceae comprises several genera, which are widely exploited for their medicinal, pharmaceutical and ornamental potential. In South Africa, several members of the Hyacinthaceae are harvested without permits from wi ld populations, processed and then sold as traditional medicine. This is reducing the den­sity, distribution and genetic divers ity of wild populations. Fur­thermore, the enforcement of existing legislation has proved ineffective with vast quantities of plants being traded locally and internationally. Consequently, it has been suggested that ex situ

t.:onse::rvation through cultivation may alleviate pressures on natu­ral resources, whilst meeting the demand for these plants. Con­ventional vegetative propagation, however, is usually fairly s low. Micropropagation, therefore, provides a rap id and econom­ical means to propagate these endangered plants, whilst supply­ing an alternative source of superior quality plants for the consumer market.

Many bufadienolides have been isolated from members of the Hyacinthace::ae. These bufadienolides are used as cardiotonics and, therefore. have pharmaceutical potential. This has led to the identification and quantification of bufadienolides in various clones and cytotypes in vitro and in situ to select high yielding s trains suitable for commercial exploitation. In Bowiea, the same bufadienolides were accumulated at similar concentrations for both in 1·irro and in situ plants (Finnie et a/. 1994 ). In Urginea, however. the accumulation of bufadienolides was linked to the format ion of specific organs in vitro, but was independent of the genotype. explant source and regeneration system (Jha et a/. I 99 I). Micropropagation, therefore, not only provides a rapid and economical means to propagate selected chemotypes, but a lso a means to produce these bufadienolides in 1•itro. This is important economically s ince it obviates the need to cultivate these plants ex virrum.

Several members of the Hyacinthaceae are cultivated as orna­rne::ntals. although the total area allotted to the production of these bulbs is relatively small. Breeding programmes, which have been established for certain genera, may increase the popu­larity of these plants. Micropropagation, therefore, not only pro­vides a rapid and economical means to propagate newl:r-devcloped cultivars, but also a means to eliminate viruses sut.:h as Hyacinthus Mosaic Virus (B lom-Barnhoorn 1986) and Ornithogalum Mosaic Virus (Vcelar eta/. 1992), thus reducing crop losses and faci litating the export of these plants. Microprop­agation also provides a means to conserve valuable germplasm (Louw 1995), therefore retaining useful character traits for future breeding programmes.

Acknowledgements The:: National Research Foundation and the University of Natal Re::search Fund are thanked for financial assistance. Ms J.L.S. Taylor kindly provided Figures I A, F, G and H.

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