Micropropagation of members of the Hyacinthaceae with medicinal and ornamental potential-A review

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.


Introduction
The Asparngales form s 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 3 1 fami l ies including several new fam ilies. These fami lies. which include the All iaceae, Asparagaceae, Asphodelacene, Dracennaceae, Eriosperm aceae and Hyacinthaceae. compri se genera previously placed wi thi n the Liliaceae Wll.\'11 fato (Dahlgren et a/. 1985;Perry 1985). The Hyac inthaccae comprises several genera including Bo111iea. F.u~·omis, Lachenalia, Ledebouria, Scilla and l/rg inea. These genera arc charncterised by bul bs with thick and sometim es contractil e roots. basa l leaves and simple or more rarely branched racemes. The inflorescences comprise actinomorphic, bisex ual tlowers, wh ich range from w hite, blue, violet, and more rarel y yellow, red, brown to nearly black. T he Hyac inthaceae are w idely distributed, occurring in southern A frica and in a region from tht: Mediterranean to SW As ia (Dahlgren et af. 1985; Duncan & du Pless is 1989).

Medicinal a nd pharmaceutical potential
In South A frica, approximately 20 000 tons of plant material , worth about R270 million, is harvested, processed and so ld as traditional medicine annually (Gosling 1998 }. Approximately 14% of thi s plant material comprises bulbs (Mander 1997}, w hich are destru ct ive ly harvested, processed and so ld for th e treatm ent of var ious ai lments (Table I ). These bulbs, w hich are sold w hole, sliced or chopped, are usually administered as decoctions, emetics and enemas (Watt & Breyer-Brandwij k 1962; Jacot Gui llamod 197 1; Hutchings 1989; Mander et af. 1995; Wyk eta/ 1997).
$'cilia natalensis is one of the top-ten most popular med icinal plants in South Africa (Cunningham 1988;Williams 1996;Mander 1997). Despite its specially protected conse rvation 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. T his 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 pri ce of these bulbs has increased steadily wi th the decline in their avail ability and size (Cunningham 1988 /. 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 Bo wiea l'oluhi/is are thirty to sixty ti mes more active than those fi·om Digitalis (Hutchings et a/ 1996). Prosc illaridin A, w hich is derived by enzyme hydrolys is fro m sci llaren A , is produced under several trade names including Caradin®, Cardion®, Prosc illan®, Sandosc ill®, Scillac-rist®, Talusin® and Urgilan® (Budavari 1996). T hese cardiac  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 failure and certain arrhythmias (Walton eta/. 1994). 13ufadienolides such as scilliroside 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 domesti c 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 anticoagul ant-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 cy totypes 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 signifi cantly 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 ( 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 commerciali zatio n has been established over the past thirty years (N icderwieser eta/. 1998 ). The successful introducti on of these hybrids onto the international flower market could initiate other breeding programmes, increasing the popul arity 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 mu ltiplication. 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, deform ed or mutated shoots, the frequency of plantlets successfully acclimatized, and the propagation potential within a specified tim e-frame are seldom reported. These details, which should be included routinely in micropropagation reports, are essential for comm ercial fac ilities.

Axillary shoots
In Eucomis. axi llary shoots were proliferated from twin-scale explants, whi ch were cultured on medium containing combinations of BA and NAA. These shoots, which were then sub-cultured onto the same medium, proliferated more shoots resulti ng 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-stab le, 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 (H ussey 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 Hyaci nthaceae, adventitious shoots were initiated directly on, or along the periphery of, various     (30000) cxplants intluenced shoot initiati on, with young ex plants producing more shoots than old explants. This may have been linked to endogenous cytokini n levels particularl y in the intermediate and old explants (N iedcrwieser & Van Staden 1990a). In Ornithogalum, the orientation of the explants also influenced shoot initiation with <~p olar explants prod ucing 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. additi onal wounding on the surface of the explant inhibited shoot initiation (Landby & Niederwieser 1992). In I ~mcimlws. th e explant size also influenced shoot initiation, which decreased linearly with a decrease in explant size (Pierik & Post 1975). These adventiti ous 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 in itiated 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 cx plants. In IJ111riea. the shoots were initiated on the adaxial su rfaces of bul b-scales. Thi s 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 bodi es on the adaxial surfaces (Van Stadcn et a/ 199 1 ).
Some adventitious shoots have been initiated on med ium containing no plant growth regulators, although most adventiti ous 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 cyto-I ..inin-like activity (N iederwieser & Van Staden 1992). Furthermore, the substitution of cytokin in glucosides ([3G)B A, (7G)BA & (9G] I3A l for BA inhibited shoot initiation (Van Staden & Drewes 1994 ). In f/yacinthus, the addition of cytokin ins had no influence on shoot initiation. although th e addition of auxins, particularl:--IAA and IBA. increased shoot in itiation (Pierik & Steegmans 1975: Bach & Cecot 1988. In Scilla, the add ition 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 for mation (Peck & Cumming 1986), while i n/~\'(ld/1/h us . the add ition ofpaclobutrazo l promoted bulblet for mation (Bach el a/ 1992). Some adventitious shoots produced roots and/or bul bs spontaneously. Other adventitious shoots have been rooted on med ium 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. Although adventitious shoots arc genetically less stable than ax illary shoots, particularly when they originate from si ngle cells, (Hussey 1980) this technique is frequently used to clone medicinal and ornamental plants rapidly and economi cally.

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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 med ium containing low concentrations of 2,4-D (Jha & Sen 1985). In Omithogalum. callus was initiated on medium contai ning 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 call us was comprised of a mixture of diploid and tetraploid cells. He al so fo und 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 regul ators (Hussey 1976b) or medium con taining combinations of BA and NAA (Nayak & Sen 1995). In Urginea, callus was initiated on medi um containing 2,4-D, NA A 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 concentrati ons of auxins and vitam ins (J ha & Sen 1984).
Although vast quantities of adventitious shoots can be produced from callus, the frequency of genetically-aberrant shoots may be rel atively high. Thus, propagation via indirect adventitious shoots is usually avoid ed . In certain breeding programmes, however, genetically-aberrant plantlets could be a useful so urce of somaclonal variation .

Direct and indirect somatic embryos
In Scilla, embryogenic callus was initiated on mediu m containing NAA and coconut mil k. The embryogen ic callus, which was comprised of small thin -walled cells with large nuclei, formed shoots and roots when transferred to medium contai ning no plant growth regulators (Chakravarty & Sen 1989). In Urginea. embryogenic and non-embryogenic callus was in itiated on medium containing 2,4-D and coconut milk. The embryogenic callus, wh ich 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 containi ng kinetin and NAA. In Urginea, the age of the callus, the frequency of sub-culturing and the cytological state ofthe callus influenced the initiati on and subsequent development of the callus (Jha & Sen 1986). The ploidy of the callus (diploid & tetrap loid) also influenced medium requirements (Jha eta/. 199 1 ). Despi te the potenti al of thi s techn ique, somatic embryos have not been induced in many members of the Hyacinthaceae.

Conclusions
The Hyacinthaceae comprises several genera, which are wide ly exploited for their medici nal, pharmaceutical and ornamental potential. In South Africa, several members of the Hyacinthaceae are harvested without permits fro m wi ld popu lations, processed and then sold as traditional med icine. This is reducing the density, distribution and ge netic divers ity of wild populations. Furthermore, the enforcement of ex isting legislation has proved ineffective with vast quantities of plants being traded locally and internati onally. Consequently, it has been suggested that ex situ t.:onse::rvation throug h cultivation may alleviate pressures on natural resources, whilst meeting the demand for these plants. Conventi onal 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 cardi otonics 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 si milar 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 formation 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 economi cal means to propagate selected chemotypes, but a lso a means to produce these bufadienolides in 1•itro. This is important eco nomically s ince it obviates the need to cultivate these plants ex virrum.
Several members of the Hyacinthaceae are cultivated as ornarne::ntals. althoug h the total area allotted to the production of these bulbs is relatively sma ll. Breeding programmes, which have been establis hed for certain genera, may increase the popularity of these plants. Micropropagation, therefore, not on ly prov ides a rapid and economical means to propagate newl:r-devcloped cultivars, but also a means to elim inate viruses sut.:h as Hyacinthus Mosaic Virus (B lom-Barnhoorn 1986) and Ornit hogalum 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.