c07_1 10/08/2007 417 7 In Vitro Storage Organ Formation of Ornamental Geophytes Glendon D. Ascough and Johannes van Staden Research Centre for Plant Growth and Development School of Biological and Conservation Sciences University of KwaZuluNatal, Pietermaritzburg, Private Bag X01 Scottsville, 3209 South Africa John E. Erwin Department of Horticultural Science University of Minnesota 1970 Folwell Avenue St. Paul, MN 55108 USA I. INTRODUCTION II. FACTORS AFFECTING IN VITRO PRODUCTION OF STORAGE ORGANS A. Explant Type and Orientation B. Temperature C. Photoperiod D. Light Quality E. Carbohydrates F. Activated Charcoal G. Plant Growth Regulators H. Gelling Agent III. DORMANCY AND ASSIMILATE ACCUMULATION OF IN VITRO–PRODUCED STORAGE ORGANS IV. MASS PROPAGATION OF STORAGE ORGANS USING LIQUID CULTURE V. FUTURE AREAS OF RESEARCH VI. ACKNOWLEDGMENTS VII. LITERATURE CITED Abbreviations ABAAbscisic acid ACActivated charcoal Horticultural Reviews, Volume 34, Edited by Jules Janick ISBN 9780470171530 ß 2008 John Wiley & Sons, Inc. 417 c07_1 10/08/2007 418 418 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN B-9Daminozide BA Benzyladenine cAMPCyclic adenosine monophosphate GAGibberellin IAAIndoleacetic acid IBAIndolebutyric acid iPIsopentenyladenine LDLong day NAANaphthaleneacetic acid PACPaclobutrazol QTLQuantitative trait locus SDShort day I. INTRODUCTION The first report of in vitro bulb production of an ornamental geophyte was for Lilium speciosum by Robb in 1957. Since then, storage organs from many species have been produced in tissue culture (Table 7.1). Transferring in vitro propagated plantlets to the ex vitro environment is a critical part of the propagation process. If this stage is successful, financial losses can be minimized. However, substantial numbers of micropropagated plants do not survive the transfer from the in vitro environment to the greenhouse. This occurs because the greenhouse has considerably lower relative humidity, higher light levels, and septic conditions that are hazardous to micropropagated plants compared to the in vitro environment. Traditionally, in vitro shoots are transferred to a rooting medium and then planted into a high-humidity environment for acclimatization. This is necessary because in vitro plantlets are not autotrophic (McCartan et al. 2004), often lack a functional cuticle, and have impaired stomatal functioning (Preece and Sutter 1991; Hazarika 2006). It is possible to reduce or eliminate this problem by inducing shoots to form a storage organ in vitro. These organs are usually resilient and can be stored or planted when desired. Other advantages of stimulating storage organ formation include: (1) elimination of in vitro rooting, (2) prevention of hyperhydricity that can result from high multiplication rates, (3) increased survival rates, and (4) a shorter bulb production period (Kim and De Hertogh 1997). Formation of storage organs in vitro can also provide a means of producing valuable medicinal compounds that only accumulate in specific storage organs (Jha et al. 1991). Geophytes produce several different types of storage organs. Functionally, these organs are very similar since they store carbohydrates, nutrients, and other components, but they differ structurally. This is c07_1 10/08/2007 Table 7.1. Summary of successful in vitro induction of storage organs of ornamental geophytes. Explant Type Factors Investigated References Alliaceae Allium ampeloprasum (B)z Ipheion uniflorum (B) Peduncle Bulb sections Hx H Ziv and Lilien-Kipnis 2000 Hussey 1975 Alstroemeriaceae Alstroemeria ‘Sweet Laura’ (R) Rhizome H Chiari and Bridgen 2000 Amaryllidaceae Amaryllis belladonna (B) Crinum ‘Ellen Bosanquet’ (B) Crinum macowanii (B) Crinum moorei (B) Crinum variabile (B) Cyrtanthus clavatus (B) Cyrtanthus loddigesianus (B) Cyrtanthus speciosus (B) Cyrtanthus spiralis (B) Eucrosia radiata (B) Eucrosia stricklandii (B) Galanthus nivalis (B) Galanthus elwesi (B) Haemanthus coccineus (B) Hippeastrum ‘Hermitage’ (B) BS, TSy Tri-scales, Sh TS TS TS TS TS TS TS Peduncle TS BS BS Peduncle Bulb sections H H, L H, T AC, C, H, L, T AC C, M C, H, M C, H, M C, M AC, H AC, C, M AC, C, H, M AC, C, H, M H H Bulb sections BS H, M H, BS TS H C, H De Bruyn et al. 1992 Ulrich et al. 1999 Slabbert et al. 1993 Fennell 2002 Fennell et al. 2001 Morán et al. 2003 Angulo et al. 2003 Angulo et al. 2003 Morán et al. 2003 Ziv and Lilien-Kipnis 2000 Colque et al. 2002 Staikidou et al. 2006 Staikidou et al. 2006 Ziv and Lilien-Kipnis 2000 Hussey 1975; Scholten and Pierek 1998; Huang et al. 2005 Ilczuk et al. 2005 Yanagawa and Sakanishi 1980 Kromer 1985 Yanagawa and Sakanishi 1980 Santos et al. 2002 (Continues) Hippeastrum x chmielii (B) Hymenocallis (B) Leucojum (B) Lycoris (B) Narcissus asturiensis (B) 419 419 Family and Species c07_1 10/08/2007 420 Table 7.1. (Continued) Explant Type Factors Investigated References Narcissus Narcissus Narcissus Narcissus Bulb Shoot clumps Bulb sections TS, peduncle H C, H H AC, H, L Narcissus triandrus (B) Nerine x Mansellii (B) Nerine bowdenii (B) Nerine sarniensis (B) Sternbergia clusiana (B) Sternbergia fischeriana (B) Shoot clumps SE, pedicels TS Peduncle BS, TS BS, embrypo H H H, seasonal, T H H H Santos et al. 1998 Chow et al. 1992 Hussey 1975 Steinitz and Yahel 1982; Ziv and Lilien-Kipnis 2000 Santos and Salema 2000 Lilien-Kipnis et al. 1992, 1994 Mochtak (1989); Jacobs et al. 1992 Ziv and Lilien-Kipnis 2000 Oran and Fattash 2005 Mirici et al. 2005 Apocynaceae Ceropegia bulbosa (T) Ceropegia jainii (T) Nodes Nodes H H Patil 1998 Patil 1998 Aracaceae Taro (Colocasia esculenta) (C) Zantedeschia jucunda (T) Apical bud Plantlets C, H L Zhou et al. 1999 Jao et al. 2005 Basellaceae Ullucus tuberosus (T) Tuber H Jordan et al. 2002 Colchicaceae Gloriosa superba (C) Corm buds, Sh C, H Sandersonia aurantiaca (C) Iphigenia indica (C) Sh Corm C H Finnie and van Staden 1989; Sivakumar et al. 2003 Finnie and van Staden 1989 Mukhopadhyay et al. 2002 Droseraceae Drosera peltata(T) Sh H, M, pH Kim and Jang 2004 bulbocodium (B) jonquilla (B) pseudonarcissus (B) tazetta (B) Hyacinthaceae 420 Family and Species c07_1 H H H H C, EO, H, T Lachenalia ‘Romelia’, ‘Ronina’, ‘Namakwa’ (B) Merwilla (formerly Scilla) (B) Merwilla natalensis (B) Merwilla sibirica (B) Muscari (B) Muscari armeniacum (B) Muscari botryoides (B) Sh C, L, T BS Sh Bulb sections H Ac, agar, C H BS BS AC H Muscari racemosum (B) Ornithogalum dubium (B) BS Peduncle H, L H Ornithogalum longibracteatum (B) Ornithogalum thyrsoides (B) Urginea indica (B) Urginea maritima (B) Bulb Bulb Bulb Bulb H H H H Iridaceae Crocus sativus (C) Corm H, L, T Sh Sh, Buds AC, C C, H Corm H, S, T Dierama luteoalbidum (C) Gladiolus ‘Friendship’, ‘Her Majesty’, ‘American Beauty’ (C) Gladiolus dalenii (C) sections sections sections sections Hannweg et al. 1996 Kongbangkerd et al. 2005 Ngugi et al. 1998 Yanagawa and Sakanishi 1980 Saniewski et al. 1974; Pierik and Steegmans 1975; Kim et al. 1981; Bach et al. 1992a, b; Bach and Swiderski 2000; Ziv and Lilien-Kipnis 2000; Yi et al. 2002 Slabbert and Niederwieser 1999 Yanagawa and Sakanishi 1980 McCartan 1999 Hussey 1975 Kromer 1985 Peck and Cumming 1986 Hussey 1975; Kromer and Kukulczanka 1992 Kromer 1989 Yanagawa and Sakanishi 1980; Ziv and Lilien-Kipnis 2000 Malabadi and van Staden 2004 Hussey 1976a Jha et al. 1984; Jha et al. 1991 El Grari and Backhaus 1987 Ilahi et al. 1987; Milyaeva et al. 1995; Piqueras et al. 1999 Madubanya 2004 Dantu and Bhojwani 1987; Ziv 1989; Ziv et al. 1998 De Bruyn and Ferreira 1992 (Continues) 421 IS Bulb sections BS BS Leaf, BS 10/08/2007 421 Bowiea volubilis (B) Charybdis numidica (B) Drimia robusta (B) Galtonia (B) Hyacinthus orientalis ‘Delft’s Blue’, ‘Carnegie’, ‘Pink Pearl’ (B) c07_1 422 Explant Type Factors Investigated References Gladiolus grandiflorus (C) Gladiolus grandzjlorus (C) Gladiolus nanus (C) Gladiolus tristis (C) Gladiolus x Homoglossum (C) Iris hollandica (B) Ixia viridifolia (C) Sparaxis hybrids (C) Watsonia vanderspuyiae (C) Peduncle Shoot Shoots Corm Corm plantlet Corm Seedlings Corm H H, L, T C H, S, T H H H Agar, C, T AC, agar, C, T, Ziv and Lilien-Kipnis 2000 Nhut et al. 2004 Ziv et al. 1998 De Bruyn and Ferreira 1992 Sutter 1986 Hussey 1976b Sutter 1986 Hauser and Horn 1991 Ascough et al. 2006 Bulb sections TS BS IS, BS, Sh C H H, L, M, T Agar, H, M Lilium auratum (B) Shoot clumps H Lilium candidum (B) Lilium japonicum (B) Lilium longiflorum (B) BS, leaf BS BS, Sh H H, L, T C, EO, H, L Lilium martagon (B) Lilium regale (B) BS Anther filament H H Lilium rubellum (B) Leaves C, H, L Hou et al. 1997 Kukulczanka et al. 1989 Paek and Murthy 2002 Kim et al. 1994; Nhut 1998; Scholten and Pierik 1998; Marinangeli et al. 1998; Lim et al. 1998; Seon et al. 2000; Lian et al. 2003 Niimi and Onozawa 1979; Takayama and Misawa 1982/1983 Sevimay et al. 2005 Maesato et al. 1994 Leshem et al. 1982; Takayama and Misawa 1982/1983 Rybczynski and Gomolinska 1989 Montezuma-de-Carvalho and Guimaraes 1974 Niimi and Onozawa 1979 Liliaceae Calochortus nuttallii (B) Fritillaria meleagris (B) Fritillaria thunbergii (B) Lilium ‘Pesaro’, ‘Dame Blanche’, ‘Casablanca’, ‘Cherry Blossom’, ‘Acapulco’ (B) 422 Family and Species 10/08/2007 Table 7.1. (Continued) c07_1 H, seasonal Lilium x formolongi (B) Tulipa ‘Merry Widow’ (B) Tulipa batalinii (B) Tulipa eichleri (B) Tulipa gesneriana (B) Seedlings IS Bulb sections Bulb sections IS H C, H, T H H T Robb 1957; Takayama and Misawa 1982/1983; Aguettaz et al. 1990; Delvallée et al. 1990; de Klerk et al. 1992 Shimasaki and Fukumoto 2000 Rice et al. 1983 Hussey 1975 Hussey 1975 Chanteloube et al. 1995 Orchidaceae Pterostylis sanguinea (T) Geodorum densiflorum (pB) Otochilus alba (pB) Seedling Rhizome Apex C, H, seasonal H H, L Debeljak et al. 2002 Roy and Banerjee 2002 Mukhopadhyay and Roy 1994 Primulaceae Cyclamen persicum (eH) Microshoots C, H Karam and Al-Majathoub 2000 Ranunculaceae Anemone coronaria hybrids (T) Plantlets H Ruffoni et al. 2005 Themidaceae Dichelostemma cingestum (C) Triteleia ixioides (C) Triteleia laxa (C) Corm sections Corm sections Corm sections H, S H, S H, S Ilan et al. 1995 Ilan et al. 1995 Ilan et al. 1995 z B ¼ bulb; C ¼ corm; eH ¼ enlarged hypocotyl; pB ¼ pseudobulb; R ¼ Rhizome; T ¼ tuber y BS ¼ bulb scales; IS ¼ inflorescence stem; TS ¼ Twin scales; SE ¼ somatic embryos; Sh ¼ shoots x AC ¼ activated charcoal; C ¼ carbohydrates; EO ¼ explant orientation; H ¼ hormones; L ¼ light/dark; M ¼ media type (liquid vs solid) or composition; T ¼ temperature 423 BS 10/08/2007 Lilium speciosum (B) 423 c07_1 10/08/2007 424 424 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN because these organs are derived and/or modified from different preexisting organs. In a review of this nature, it may be helpful to define each of these types of storage organ in terms of their morphological origin. A bulb is a specialized underground organ that contains a vertical stem axis with an apical meristem or flower primordium with a series of leafy scales that adhere closely. The Hippeastrum bulb type is the simplest and is composed entirely of leaf bases. The tulip bulb type, in contrast, is composed entirely of concentric scales separated by very short internodes, while the narcissus type is made up of both leaves and scales (Rees, 1972). A corm is a vertical, swollen underground base of a stem axis that is typically surrounded by protective skins or tunics, and is a solid structure with distinct nodes and internodes. A tuber is a swollen underground stem, usually formed on a stolon. Tuberous roots are thickened underground storage structures that are derived from roots. Although the storage organs of begonia, cyclamen, and gloxinia are often described as tubers, they are in fact modified hypocotyls. A pseudobulb is the thickened stem of a sympodial orchid that has waterstorage capacity. A rhizome is a thick, horizontal underground stem with nodes and scalelike leaves; roots form on the lower surface and new shoots form at nodes. While all these structures store carbohydrates, water, nutrients, and other components, their structure and origin of formation differ greatly. Thus, inductive conditions are often not the same (Krikorian and Kann 1986). Although various reviews have covered the physiology, tissue culture, and biotechnology of ornamentals (De Hertogh and Le Nard 1993; Kim and De Hertogh 1997; Fennell and van Staden 2004), the in vitro induction of storage organs has not been covered. This review: (1) summarizes procedures for producing storage organs in vitro; (2) covers differences and commonalities in requirements among species, genera, and families; and (3) provides areas for future investigations. II. FACTORS AFFECTING IN VITRO PRODUCTION OF STORAGE ORGANS The in vitro environment is ideal for plants in that it contains all the necessary requirements needed for optimal growth. These include: water, macro- and microelements, light, and an unlimited supply of carbohydrates. Additionally, in vitro grown plantlets are not exposed to external stresses, such as competition for space and nutrients, herbivory, insect attack, pathogen and disease pressure, temperature extremes, flooding, or drought. These superlative conditions, while c07_1 10/08/2007 425 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 425 beneficial for rapid growth, may not necessarily be optimal for induction of storage organs, since they are adaptations to ensure survival during adverse environmental conditions. Thus, media composition is altered by addition of plant growth regulators or adjusting carbohydrate levels, or cultural conditions are changed to mimic those of the external environment and so induce the formation of storage organs. A. Explant Type and Orientation Choosing the correct explant is essential if the desired outcome of any tissue culture procedure is to be achieved with minimal delays. The type of explant for inducing a storage organ varies by family (Table 7.1). For example, Liliaceae and Hyacinthaceae form adventitious bulbs readily when bulb scale, twin-scale, or leaf explants are used (van der Linde 1992). In the Amaryllidaceae, twin-scale and bulb scale explants are commonly used, but no report of successful induction from leaf explants has been found. In contrast, shoot or corm explants are more frequently used for corm-producing geophytes of the Colchicaceae and Iridaceae (Finnie and van Staden 1989; Ziv 1989; Madubanya 2004). Twin-scale explants are comprised of two scales with a portion of the base plate between them. The technique utilizes the same principle as that for commercial twin scaling, where a band of undamaged totipotent cells at the base of the scales differentiate to produce bulbils (Rees, 1992). The use of individual bulb scales or leaves allows more explants to be taken from the same parent, thereby increasing production of propagules. This method relies on dedifferentiation of existing specialized cells to attain totipotency, followed by redifferentiation and subsequent bulblet formation. This is termed adventitious bulblet formation since the storage organs are produced de novo. For plants that produce tubers, rhizomes, or corms, all that is required is modification of an existing organ. The literature shows a wide variety of explants being used for the induction of storage organs in vitro. Kim et al. (1981) used excised flower buds as explants to initiate bulbs of Hyacinthus orientalis ‘Anna Marie’ and ‘Delft Blue’. The inflorescence stalk of Bowiea volubilis is also capable of regenerating bulbs (Hannweg et al. 1996). Anther filaments of Lilium regale form bulblets through indirect organogenesis (Montezuma-de-Carvalho and Guimaraes 1974). Even microspores are capable of producing bulblets (Sharp et al. 1971). Stem nodes have produced nondormant pseudobulblets in Lilium longiflorum cultures (Nhut 1998), and Mirici et al. (2005) used immature embryos of Sternbergia c07_1 10/08/2007 426 426 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN fischeriana to produce bulblets. Shoot explants have been extensively used for corm induction in the Iridaceous Gladiolus cv. ‘Eurovision’ (Ziv 1989), Dierama luteoalbidum (Madubanya 2004) and Watsonia vanderspuyiae (Ascough et al. 2006). Jacobs et al. (1992) found that if twin-scale explants of Nerine bowdenii were placed on medium in an apolar orientation, the percentage of explants that produced bulbs increased from 15% to 79%. The average number of bulblets produced per explant were comparable for polarly and apolarly orientated explants, but the average fresh weight per bulblet in apolar explants was more than double those of polar explants. Fennell (2002) investigated the effect of explant age on bulblet induction in Crinum moorei and showed that twin-scale explants from the middle section responded better than those excised from the inner (younger) or outer (older) part of the bulb. These explants produced more bulblets that were of a greater diameter than those excised from the inner or outer part of the bulb. In contrast, Robb (1957) found no difference in the ability of scale explants of different ages to produce bulblets in Lilium speciosum. Both single-scale and twin-scale explants have been used to induce bulbs in Sternbergia clusiana (Oran and Fattash 2005). Although the frequency was only 0.73 bulbs per explant for both single scales and twin scales, these frequencies occurred at different hormone combinations. Lilium bulbs are only initiated on the adaxial surface of bulb-scale sections, irrespective of the orientation of the explant or species (Robb 1957; Leshem et al. 1982). Although bulblets can be regenerated indirectly via an intermediate callus phase from various types of explants, scale explants will generally produce more bulbs (Lesham et al. 1982). The choice of a storage organ for an explant type is unfortunately destructive since the parent plant will be used. This may pose a problem if rare or endangered species need to be cultured in vitro for the purpose of conservation. In addition, the leaf explants of many species, especially Iridaceae, do not respond adequately to in vitro techniques. To some extent, these difficulties may be overcome by using explants derived from the inflorescence. Ziv and Lilien-Kipnis (2000) succeeded in propagating and subsequently inducing storage organ formation on inflorescence explants from nine species belonging to four families (Alliaceae, Amaryllidaceae, Hyacinthaceae and Iridaceae). This broad response across the different families is encouraging, although more genera and families need to be tested. Indirect formation of bulbs through a callus stage is also a possibility, and in this procedure, auxins are used to initiate callus on the explants. This has been successful in several species, including Nerine bowdenii c07_1 10/08/2007 427 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 427 (Jacobs et al. 1992), Lilium longiflorum (Sheridan 1968), and Ornithogalum longibracteatum (Malabadi and van Staden 2004). After callus is obtained, cytokinins (Sivakumar et al. 2003) are used to regenerate the appropriate storage organ. Perhaps one disadvantage of this technique is the potential for the introduction of undesired somaclonal variation that often arises during indirect organogenesis. In banana, a routinely in vitro–propagated commercial crop, somaclonal variation may occur in as many as 70% of explants (Bairu et al. 2006). However, variations induced through in vitro culture methods may be a useful tool for introducing novel plants (Debergh 1994). The timing of explant selection also plays a role in determining the success of storage organ induction (Debeljak et al. 2002). For example, Lilium speciosum explants produced bulblets only during autumn and spring, suggesting that regeneration of in vitro bulbs is limited to periods of vegetative growth (Robb 1957). Similar results were found by Niimi and Onozawa (1979), who utilized leaf explants taken after plants had flowered. In the case of ornamentals such as Cyclamen that have storage organs derived from enlarged hypocotyls, root explants are generally more effective than shoot explants for storage organ formation (Karam and Al-Majathoub 2000). In contrast, rhizome explants were more effective at forming pseudobulbs in the orchid Geodorum densiflorum (Roy and Banerjee 2002). B. Temperature Temperature regulates not only growth rates but also the transition between various vegetative and reproductive phases during development. In many temperate ornamental geophytes, reduced temperatures simulate the onset of winter during which many species enter ‘‘dormancy’’ (Rees 1992; Le Nard and De Hertogh 1993). Low in vitro temperatures may also simulate this natural seasonal change to induce storage organs capable of enduring unfavorable growth conditions. This is evident in the literature, as nearly all of the species and genera (except Crinum) respond well to reduced culture temperatures. Nerine bowdenii bulbs were readily formed at 128 to 228C, but at higher temperatures (278C), bulblet formation decreased (Jacobs et al. 1992). Exposing Hyacinthus orientalis cv. ‘Carnegie’ leaf explants for 12 to 28 weeks to 48C and subsequently culturing them at 238C increased bulblet induction as compared to explants grown at 238 or 48C only (Bach et al. 1992a). A three-month 58C treatment followed by culture at 208C increased bulblet production in Tulipa gesneriana cv. ‘Lucky c07_1 10/08/2007 428 428 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN Strike’ compared to explants that were treated for one month at reduced temperatures (Chanteloube et al. 1995). Similar results were reported for Lachenalia varieties ‘Romelia’, ‘Ronina’, and ‘Namakwa’, another genus of Hyacinthaceae (Slabbert and Niederwieser 1999). In these cultivars, low temperatures of 48, 108, or 158C induced bulblet formation on shoots that were at least 4 millimeters (mm) long. However, when temperature was 218C, bulb formation was inhibited. When shoots were less than 4 mm, bulbs were not formed under inductive conditions. This suggests that a specific developmental (physiological) status must be attained before bulbs can be formed. Genera from the Iridaceae that form corms require different temperatures for induction. In Crocus sativus, corm formation was promoted at 108C (Milyaeva et al. 1995). Within a genus, temperature requirements can differ. For example, Gladiolus dalenii formed corms at 258C while G. tristis required 158C to induce corming (De Bruyn and Ferreira 1992). This difference in temperature requirement could result from the different natural habitats; that is, G. dalenii occurs in a summer rainfall area but G. trisits occurs in a winter rainfall area. In another species, G. grandzjlorus, corm formation was promoted at 158 and 208C but corms were inhibited from forming at 258C (Nhut et al. 2004). In Watsonia gladioloides, W. laccata, and W. lepida, corms did not form after 3 months at 108, 158, 208, or 258C, but successful corm induction only occurred in W. vanderspuyiae at 258C (Ascough et al. 2006). In several unnamed Sparaxis hybrids, cultures grown at 158C had a higher corm induction frequency compared to those grown at 208C (Hauser and Horn 1991). In contrast, a temperature of 158C prevented bulbs from forming on explants of Crinum macowanii (Slabbert et al. 1993) and C. moorei (Fennell 2002). For C. macowanii, optimal induction occurred at 258 and 308C, but bulblet formation in C. moorei was strongly inhibited at 308C. Similarly, reducing culture temperature in Calochortus nuttallii did not increase bulb formation (Hou et al. 1997). C. Photoperiod Ornamental geophyte responses to photoperiod in tissue culture vary greatly. Plants that require light to form storage organs in vitro include: Nerine bowdenii (Jacobs et al. 1992), Lilium oriental hybrid ‘Casablanca’ (Lian et al. 2003), and Fritillaria thunbergii (Paek and Murthy 2002). In these species, continuous darkness inhibited storage organ formation, but a 16-hour (h) photoperiod promoted in vitro storage organ formation. Other species produced in vitro storage organs better in the dark: c07_1 10/08/2007 429 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 429 Narcissus tazetta (Steinitz and Yahel 1982), Lilium rubellum (Niimi and Onozawa 1979), and Lilium longiflorum ‘SnowQueen’(Marinangeli et al. 1998). Species that produced bulbs equally in the light or dark include Hyacinthus orientalis cv.s ‘Anna Maria’ and ‘Delft Blue’ (Kim et al. 1981), Lilium longiflorum cv. ‘Osnat’ (Leshem et al. 1982), and Lachenalia cv.s ‘Romelia’, ‘Ronina’, and ‘Namakwa’ (Slabbert and Niederwieser 1999). The mechanism of photoperiod-induced storage organ formation is unknown, although one may tentatively speculate that it is at least in part regulated by phytochrome. In a natural environment, a change in photoperiod would herald the start of a new season, and this could promote induction of storage organ formation as a means to survive the oncoming adverse conditions, such as extreme temperatures or dry winters or summers. This is the case for the ex vitro production of storage organs (Rees 1992) where, in most species, the promotion of storage organ formation by SD is accompanied by reduced aerial growth followed by dormancy induction. Two notable exceptions are the essential requirement of Allium for LD for bulb formation, and the suppression of rhizome formation under SD in Alstroemeria. D. Light Quality In Hyacinthus orientalis, Bach and Swiderski (2000) found that blue light (480 nm) stimulated adventitious shoot production, while complete darkness, red (680 nanometer [nm]) or white light (from standard fluorescent tubes) promoted bulb formation. In Solanum tuberosum, a model system for tuberization, Goeden and Tong (2003) showed that darkness, white light, red light, and far-red light (730 nm) stimulated tuber formation in ‘Desirée’ and ‘Norland’, but blue light inhibited tuberization in ‘Norland’ but not ‘Desirée’. This suggested the blue light receptor cryptochrome may be involved in regulating tuber induction. Despite excellent advances in the understanding of storage organ formation in potato, very little work has been performed on ornamental geophytes. We are still very much in the dark with respect to the effect of light quality on the induction of storage organs in vitro, and there is scope for further research in this area. E. Carbohydrates Carbohydrates are the main source of energy for respiration and structural components, especially the cell wall. Since storage organs enable the plant to endure unfavorable environmental conditions by sequestering water, carbohydrates, proteins, and lipids, it seems logical to assume that c07_1 10/08/2007 430 430 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN carbohydrate supply will play a major role in both the induction and subsequent growth of storage organs. Sucrose is the most prevalent carbohydrate used for micropropagation and is commonly added at a concentration of 3% (weight/volu mew/v). For several species, this concentration is sufficient for storage organ induction. In the oriental Lilium hybrids ‘Pesaro’, ‘Dame Blanche’, ‘Casablanca’, ‘Cherry Blossom’, and ‘Acapulco’, Lim et al. (1998) found that 3% sucrose in the culture medium was optimal for producing bulbs, and while increasing the sucrose content decreased the number of bulbs formed, it increased the average bulb weight. This also occurred in Lilium rubellum (Niimi and Onozawa 1979). Differences have been reported for different Lilium species; for example, L. rubellum produced more bulbs at 4% sucrose, while L. longiflorum required 2% sucrose (Nhut 1998). Bach et al. (1992b) examined the effects of sucrose, fructose, and glucose on bulblet induction on Hyacinthus oreintalis ‘Delft’s Blue’ leaf explants and found that media containing fructose produced more bulblets. Interestingly, the three carbohydrates all induced significantly more bulblets when supplied at 3% as compared to a 6% concentration (Bach et al. 1992b). However, Bach and Swiderski (2000) found that 13% glucose concentration was optimal for regenerating Hyacinthus orientalis bulbs. In Lachenalia cultivars ‘Romelia’, ‘Ronina’, and ‘Namkawa’, no difference in bulb production was found between 3% and 6% sucrose concentration; however, increased sucrose concentrations increased average bulb diameter (Slabbert and Niederwieser 1998). Similarly, no significant differences were found in the number of bulbs produced on 3% or 9% sucrose concentrations for Cyrtanthus loddigesianus and C. speciousus (Anguloet al. 2003) Optimum sugar concentrations for storage organ formation can vary for cultivars within a genus. For example, Dantu and Bhojwani (1987) found that Gladiolus ‘Her Majesty’ and ‘American Beauty’ formed corms optimally at 10% sucrose, while ‘Friendship’ optimally formed corms in 6% sucrose. Formation of Narcissus jonquilla bulblets was enhanced when sucrose levels were increased to 6% or 9% (Chow et al. 1992), while 9.5% sucrose was used for Eucrosia stricklandii (Colque et al, 2002) and Calochortus nuttallii (Hou et al. 1997). For Cyrtanthus clavatus and C. spiralis, 9% was optimum (Morán et al. 2003). For Crinum moorei, a 6% sucrose concentration improved bulb production compared to 4% or 8% (Fennell 2002). Low and intermediate sucrose levels (3% or 6%, respectively) induced enlarged hypocotyl formation in Cyclamen, but higher concentrations (9% or 12%) suppressed ‘‘tuberization’’ (Karam and Al-Majathoub 2000). c07_1 10/08/2007 431 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 431 The mode of action of carbohydrates has not been definitively shown and may differ with the genus and species. Increasing medium soluble carbohydrate levels increases the medium osmolarity. How ever, when the osmolytes mannitol or sorbitol were added to the medium at similar concentrations, they did not stimulate bulbing of Narcissus ‘St. Keverne’ and ‘Hawera’ (Staikidou et al. 2005). This suggests that carbohydrate supply is more important than osmolarity for bulblet development. F. Activated Charcoal Activated charcoal (AC) is added to cultures for several functions, including: (1) adsorption of undesirable or inhibitory substances, (2) providing a dark environment to allow root production, and (3) adsorption and stabilization of isotherms and plant growth regulators. It may also release compounds into the culture medium (Pan and van Staden 1998). Addition of AC to the growth medium increased bulblet production in Narcissus tazetta (Steinitz and Yahel 1982). The use of AC overcame the inhibition of bulblet formation by NAA and BA. AC also enhanced bulb formation in Muscari armeniacum ‘Early Giant’ scale explants with no intermediate callus stage (Peck and Cumming 1986). However, addition of AC did not increase bulb formation in Calochortus nuttalli (Hou et al. 1997), and it inhibited corm formation in Dierama luteoalbidum at high (8%) sucrose concentrations (Madubanya 2004). Addition of AC to the culture medium more than doubled bulblet regeneration in Crinum variabile (Fennell et al. 2001), but it did not influence bulblet induction in C. moorei (Fennell 2002). In Lilium longiflorum ‘Georgia’ cultures, AC increased number of bulbs per shoot, bulb diameter, as well as average fresh weight of the bulblet (Han et al. 2004). Whether these effects are due to its adsorptive capability, release of promotive substances, or simply providing a dark environment remains unknown. G. Plant Growth Regulators Plant growth regulators are involved in nearly all aspects of plant growth and development, and the induction of in vitro storage organs is no exception. A direct relationship between auxin (IBA) concentration and bulb size, fresh weight, and dry weight was found in Muscari botryoides (Kromer and Kukułczanka 1992). Although the addition of a cytokinin (BA) increased the number of bulbs formed, it inhibited bulb growth and root formation. Chow et al. (1992) repeated these experiments c07_1 10/08/2007 432 432 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN on Narcissus jonquilla and found the same results. Auxin (IBA) and cytokinin (BA) were required to induce bulblet formation in Sternbergia clusiana (Oran and Fattash 2005). Although hormones added to culture media can enhance bulblet formation, they can also result in formation of abnormal bulblets (Lian et al. 2003). The use of specific types of auxins and cytokinins is often critical. For example, Maesato et al. (1994) showed that NAA in combination with iP improved response in Lilium japonicum compared to other cytokinins. This combination also promoted pseudobulb production in the orchid Otochilus alba (Mukhopadhyay and Roy 1994). In contrast with the orchid Geodorum densiflorum, BA inhibited pseudobulb development (Roy and Banerjee 2002). Using Hyacinthus orientalis ‘Pink Pearl’, Pierik, and Steegmans (1975) showed that bulb formation was promoted by IAA and IBA, but inhibited by high concentrations (10 milligrams/liter [mgl]-1) of NAA. In contrast, bulblet weight was unaffected by varying IAA concentrations, but decreased at high concentrations (10 mgl-1) of IBA and NAA (Pierik and Steegmans 1975). NAA also inhibited bulb formation in Calochortus nuttallii (Hou et al. 1997). NAA was essential for bulb induction from leaf explants of Lilium rubellum (Niimi and Onozawa 1979), and it enhanced corm formation in Ixia viridifolia and a Gladiolus
Homoglossum hybrid (Sutter 1986). Charybdis numidica (Kongbangkerd et al. 2005) and Nerine
Mansellii (Lilien-Kipnis et al. 1994) also produced storage organs when 0.5 mgl-1 NAA was used. In comparison, IBA has been used to induce bulbs of Hyacinthus orientalis ‘Carnegie’ (Paek and Thorpe 1983; Yi et al. 2002) and Narcissus asturiensis (Santos et al. 2002). Bulb formation in Narcissus jonquilla was inhibited by BA, while NAA had no effect on bulbing (Chow et al. 1992). The reverse was found in Hyacinthus orientalis cv.‘Pink Pearl’, where kinetin and BA did not affect bulb regeneration, but IAA, IBA, and NAA were effective (Pierik and Steegmans 1975). Microtubers formed on Ceropegia bulbosa and C. jainii explants when BA or kinetin were present in the medium; when both were used, microtuber formation doubled (Patil 1998). BA has been shown to increase bulb production in Urginea maritima (El Grari and Backhaus 1987) and Lilum longiflorum (Nhut 1998), Crinum moorei (Fennell 2002); rhizome formation in Alstroemeria ‘Sweet Laura’ (Chiari and Bridgen 2000); and tuberization in Cyclamen persicum (Karam and Al-Majathoub 2000). Interestingly, tuber production in Drosera peltata was inhibited by kinetin and BA (Kim and Jang 2004). Several cytokinins were used for different explants to induce corm formation in c07_1 10/08/2007 433 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 433 Gloriosa superba cultures (Sivakumar et al. 2003): BA was used when inducing corms from callus tissue, kinetin when the explants were nondormant corm buds, and iP when dormant corm buds were used as explants. Pretreatment by culturing explants on a medium with BA followed by culture on hormone-free medium increased bulb formation in Crinum ‘Ellen Bosanquet’ (Ulrich et al. 1999) and corm induction in Gladiolus ‘Green Bay’, ‘Wine & Roses’, ‘Top Brass’, and ‘Mornlo’ (Sen and Sen 1995). A high cytokinin: auxin ratio (10–20:1) has been used to induce bulb formation in Hyacinthus ‘Lady Derby’ (Saniewski et al. 1974) and ‘Delft’s Blue’ (Kim et al. 1981), formation of cormogenic nodules in Crocus sativus (Piqueras et al. 1999), and induced corm formation in Iphigenia indica (Mukhopadhyay et al. 2002) and to stimulate bulbing in Sternbergia fischeriana (Mirici et al. 2005), Amaryllis belladonna (De Bruyn et al. 1992), Fritillaria thunbergii (Paek and Murthy 2002), and Urginea indica (Jha et al. 1991). The reverse was found for Hippeastrum ‘Hermitage’, where a low cytokinin: auxin ratio (0.2:1) promoted bulblet formation (Huang et al. 2005), while other species, such as Bowiea volubilis (Hannweg et al. 1996) and Lilium candidum (Sevimay et al. 2005), formed bulbs when a ratio of 1 or equal amounts were present. Addition of GA3 or GA4þ7 to the culture medium repressed bulblet formation in Hyacinthus orientalis cv. ‘Pink Pearl’ (Pierik and Steegmans 1975). In contrast, a 1 mgl-1 soak in GA3 for 15 hours stimulated bulb formation in shoot explants of Tulipa ‘Merry Widow’ (Rice et al. 1983). When culture temperature was reduced from 208 to 48C, the beneficial effect of GA3 application was nullified. This suggests a temperaturehormone interaction that controls bulb formation in Tulipa. GA3 in combination with NAA and BA promoted tuber formation in Ullucus tuberosus (Jordan et al. 2002). ABA may play an important role during storage organ formation. Scale explants of Lilium speciosum ‘Rubrum’ treated with 1.32 mgl-1 ABA formed bulbs, but the addition of the ABA synthesis inhibitor fluridone (0.33 mgl-1) prevented bulb formation (Kim et al. 1994). This was verified by Shimasaki and Fukumoto (2000) who found similar results on Lilium
formolongi seedlings. In Calochortus nuttallii, the addition of ABA did not enhance bulb formation (Hou et al. 1997) while with Hyacinthus orientalis ‘Pink Pearl’ (Pierik and Steegmans 1975) and Ullucus tuberosus (Jordan et al. 2002), ABA decreased not only bulblet regeneration but also subsequent growth. The effect of ethylene has been investigated only in Hyacinthus orientalis ‘Pink Pearl’, where it decreased bulblet regeneration (Pierik c07_1 10/08/2007 434 434 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN and Steegmans 1975). This is surprising, since many auxin-regulated physiological processes in plants are mediated by ethylene. The fact that auxin is widely used for the induction of storage organs in vitro should necessitate investigations into ethylene effects. This could be studied by simultaneously adding auxin and an ethylene action or biosynthesis inhibitor to the medium and observing the subsequent growth and morphogenesis of the explants. Jasmonic acid has been implicated in promoting potato tuber development (Creelman and Mullet 1997), and bulb formation in onion and garlic (Koda 1997). High concentrations (2.2 mgl-1) reduce ethylene evolution and induce bulb formation in Lilium
formolongi seedlings (Shimasaki and Fukumoto 2000). Santos and Salema (2000) reported that Narcissus triandrous shoots formed bulbs when jasmonic acid (1 mgl-1) was combined with either NAA (0.12 mgl-1) or iP (1 mgl-1), but greatest formation of large bulbs occurred when jasmonic acid was the only hormone present. Tuberization in the terrestrial orchid Pterostylis sanguinea was promoted by jasmonic acid (Debeljak et al. 2002). However, it suppressed tuber formation in Ullucus tuberosus (Jordan et al. 2002). Traumatic acid (Marinangeli et al. 1995) and cAMP (Marinangeli et al. 1998) are two chemical messengers that induce bulbing in Lilium longiflorum ‘Snow Queen’. The addition of adenosine increased activation of adenylate cyclase, which subsequently increased intracellular cAMP levels and promoted bulblet formation (Marinangeli et al. 1998). While the authors speculate on a possible mode of action, these compounds need to be tested on more species from the same family and on species producing different types of storage organs before firm conclusions can be drawn. Some plant growth regulators retard growth by acting as anti-GA agents by preventing GA biosynthesis and reducing stem/cell elongation. Ziv (1989) successfully used daminozide (B-9), ancymidol, PAC, and uniconazole (GA biosynthesis inhibitors) to reduce shoot elongation and enhance corm production in Gladiolus ‘Eurovision’. Crinum twin-scale explants produced bulbs when 1.25 mgg-1 ancymidol was added to the medium (Slabbert et al. 1993), while PAC and imazalil have been used to increase induction and growth rate of Crocus sativus corms (Piqueras et al. 1999). Addition of PAC to shoot cultures of Dierama luteoalbidum increased corm production from 0 to 3 corms per explant (Madubanya 2004). Despite being more successful for Iridaceae, PAC did not induce corm formation in Watsonia gladioloides, W. laccata, W. lepida, or W. vanderspuyiae (Ascough et al. 2006). c07_1 10/08/2007 435 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 435 H. Gelling Agent The choice of type and quality of agar used to solidify the culture medium can influence in vitro responses. For example, Hippeastrum ‘Cinderella’ flower stem (Pierik 1991) and Hyacinthus orientalis ‘Pink Pearl’ bulb scale explants (Pierik and Ruibing 1973) were sensitive to the quality of agar brands used when the number of bulbs produced was compared. Differences were found in Lilium ‘Enchantment’ in terms of mean number of bulbs produced per explant (3.2–5.3) and average bulblet weight (120–471 mg) using seven different commercial agar brands (Scholten and Pierik 1998). In several unnamed Sparaxis hybrids, Gelrite was more effective at inducing corm formation on shoot explants as compared to agar (Hauser and Horn 1991). Investigations into bioactive compounds occurring in commercial gelling agents was undertaken by Arthur et al. (2004). They showed that several types of gelling agents (Difco Bacto Agar, Agar Commercial Gel, Agar Bacteriological and Gelrite) contain root-promoting substances that co-chromatographed with IAA. Moreover, these compounds are affected by autoclaving and interact with other additives such as AC (Arthur et al. 2006). Thus, it is critical to consider the type of gelling agent that is used during tissue culture. III. DORMANCY AND ASSIMILATE ACCUMULATION OF IN VITRO–PRODUCED STORAGE ORGANS There is often a ‘‘dormancy’’ period associated with geophytic organs produced in vitro. This may be an advantage as it allows storage and/or shipping of the propagules and also negates the need for hardening-off and acclimatization. However, a long ‘‘dormant’’ stage could cause delays in propagation or breeding programs. When produced in vitro, storage organs from some species are not dormant. Examples are: Gladiolus ‘Eurovision’ (Ziv 1989), Lilium longiflorum (Nhut 1998), Crinum moorei (Fennell et al. 2001), and C. variabile (Fennell 2002). In contrast, normally these genera have a deep dormancy that necessitates a low temperature to resume growth (Kamerbeek et al. 1970). This lack of dormancy is a beneficial by-product of the in vitro propagation system. The main factors involved in the dormancy of in vitroformed Lilium bulbs are temperature and ABA. Approximately 70% of bulbs formed at 158C were ‘‘nondormant,’’ while only approximately 5% of bulbs formed at 258C were ‘‘nondormant’’ (Aguettaz et al. 1990). If bulbs c07_1 10/08/2007 436 436 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN induced at 158C were transferred to 208 or 258C, they became ‘‘dormant’’ within six weeks (Delvalée et al. 1990). These findings suggest that lily bulbs have three phases of development: (1) bulbs that are initially nonviable and ‘‘nondormant,’’ (2) bulbs that are viable and ‘‘nondormant,’’ and (3) bulbs that are viable and ‘‘dormant.’’ This phasing into dormancy corresponds with the developmental pattern of the primordium as they cease producing leaves and start to produce scales (Delvalée et al. 1990). Adding the ABA synthesis inhibitor fluridone (0.33 mgl-1) to the culture medium resulted in bulbs being ‘‘nondormant’’ (Kim et al. 1994). ‘‘Dormancy’’ was reestablished if ABA was present in the medium. Gladiolus ‘Forest Fire’ corms require a four-week temperature treatment (28–58C) to break ‘‘dormancy’’ prior to planting out (Hussey 1977). However, if these in vitro-produced corms were subcultured onto media containing BA, ‘‘dormancy’’ was broken and leaf growth occurred. A similar period was found in other species, including Ixia viridifolia (Sutter 1986), Lilium speciosum ‘Rubrum’ (Aguettaz et al. 1990); L. auratum, L. longiflorum ‘Hinomoto’, and L. speciosum ‘Uchida’ (Takayama and Misawa 1982/1983); and Urginea maritima (El Grari and Backhaus 1987). Day length, continuous light, or continuous dark had no impact on ‘‘dormancy,’’ while GA3 partially alleviated dormancy in Lilium speciosum ‘Rubrum’ (Aguettaz et al. 1990). For Lilium longiflorum, a one-hour treatment at 458C has been used to break ‘‘dormancy’’ before being planted out (Rees 1992). Chilling has a major effect not only on meristematic regions, but also affects the physiology of the storage organ. Chilled (48C) and unchilled (178 or 208C) bulbs of Tulipa gesneriana were compared for their water mobility and carbohydrate content by Kamenetsky et al. (2003). Scales of chilled bulbs had higher water content and mobility, and increased starch degradation and accumulation of sucrose and fructans. The researchers suggested that this could result from water molecules becoming free after degradation of starch, or by an influx of free water due to the increased osmotic potential caused by the increased sugar concentrations. Little research has been carried out on changes in assimilates during storage organ formation in vitro. Yi et al. (2002) examined protein content during bulb formation in Hyacinthus orientalis ‘Carnegie’ and found that expression of several proteins was increased during culture, while others, especially a 29kD protein, were downregulated. This protein was suggested to be one of a family of major storage proteins. In most ornamental geophytes, starch and inulin are the major storage carbohydrates, but the types of proteins, amino acids, lipids, and secondary c07_1 10/08/2007 437 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 437 metabolites that are stored depends to a large degree on the species (De Hertogh and Le Nard 1993). In ex vitro grown Dahlia plants, roots accumulated fructans in response to SD treatments, with sucrose being the regulating factor for fructan metabolism (Legnani and Miller 2001). Havey et al. (2004) found a quantitative trait locus (QTL) in onion that affected soluble carbohydrates, especially fructans, in stored bulbs, suggesting that it is at least in part genetically regulated IV. MASS PROPAGATION OF STORAGE ORGANS USING LIQUID CULTURES Traditional tissue culture techniques in agar-solidified media have allowed rapid market penetration of superior genotypes by exploiting the enhanced multiplication rates resulting from an optimized protocol. Liquid culture provides an opportunity to augment this even further. In vitro propagation techniques, while more rapid, tend to be more expensive than conventional propagation methods. Mass propagation using liquid culture in a bioreactor can decrease the cost of micropropagated plantlets. While the formation of storage organs occurs in static liquid cultures (Sen and Sen 1995; Jordan et al. 2002; Paek and Murthy 2002), in some cases a static (nonagitated) medium suppresses bulb induction (Takayama and Misawa 1982/1983). Thus, the trend has been to induce bud clusters or meristemoids in bioreactors or liquid-shake cultures (Ascough and Fennell 2004) as, in addition, under these conditions the growth rate is often significantly enhanced. These clusters can be separated and placed on media for plantlet regeneration followed by storage organ induction and growth. Nerine
Mansellii bud clusters were initiated on flower pedicels in the presence of NAA, BA, and PAC and, after subculture to media with 6% sucrose and low concentrations of NAA (0.05 mgl-1), bulblets formed (Lilien-Kipnis et al. 1992, 1994). Similarly, Gladiolus ‘Eurovision’ bud explants formed proliferating bud clusters either in a liquid-shake culture (Ziv 1989) or a bioreactor (Ziv et al. 1998)containing growth retardants, and when placed on a solid medium corms were formed. The type of bioreactor is also important. Stirred bioreactors can easily damage bulblets because of the shearing forces (Lim et al. 1998). Temporary-immersion (ebb and flood) bioreactors produced more bulblets in Lilium ‘Casablanca’ when compared to suspension cultures or explants grown on solid media (Lian et al. 2003). Several researchers have used bulb sections to regenerate bulbs in Lilium c07_1 10/08/2007 438 438 G. D. ASCOUGH, J. VAN STADEN, AND J. E. ERWIN bioreactor cultures without having to subculture to another medium (L. speciosum: Takayama and Misawa 1982/1983; Lilium ‘Casablanca’: Lim et al. 1998; Seon et al. 2000; Lian et al. 2003). Other species that respond favorably to proliferation in liquid media followed by successful induction of in vitro storage organs include: Cyrtanthus loddigesianus and C. speciousus (Angulo et al. 2003); Gladiolus ‘Green Bay’, ‘Wine & Roses’, ‘Top Brass’, and ‘Mornlo’ (Sen and Sen 1995); Dichelostemma cingestum, Triteleia ixioides, and T. laxa (Ilan et al. 1995). The possibility of producing in vitro storage organs that could flower in the first season of ex vitro growth means that propagules could be marketed without growing plants in the field for an additional one to two years. This possibility of growth is species dependent, as is the timing of floral meristem initiation and inflorescence development. Takayama and Misawa (1982/1983) successfully mass-propagated three Lilium species, but only L. longiflorum flowered in the first season of growth. This was confirmed by Nhut (1998). Over 90% of in vitro formed Narcissus bulbocodium bulbs flowered in the first season of growth (Santos et al. 1998), while Cyrtanthus clavatus and C. spiralis flowered within one year of transfer to the soil (Morán et al. 2003). To date, this has been achieved only for bulbous plants. No reports have been found for other storage organ types. In addition, since flowering is often correlated with bulb size, such early flowering may not produce a salable flower/inflorescence. V. FUTURE AREAS OF RESEARCH Many questions remain about the mode of action of various inductive treatments on in vitro production of geophytic organs. Does induction by low temperature proceed in a similar manner to vernalization, where a certain minimum number of chilling hours are required? Are there species that are absolute in their requirement for a specific inductive treatment and, thus, candidates for detailed investigations? Is energy redistribution a significant factor, and if so, does affecting source/sink relationship affect the process? GA and light are critical factors in several steps of potato tuberization (Martı́nez-Garcı́a et al. 2002), but this interaction has not been explored in ornamentals. Are there specific genes that, when activated, promote or repress storage organ formation? To what extent is the morphological origin of the storage organ a factor when examining induction factors? It is clear that formation of storage organs in vitro will continue to be an important c07_1 10/08/2007 439 7. IN VITRO STORAGE ORGAN FORMATION OF ORNAMENTAL GEOPHYTES 439 part of the micropropagation process and also that a thorough understanding of this natural phenomenon will enhance optimization and increase production of propagules. VI. 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