Micropropagation and Horticultural Potential of Native Tasmanian and

Volume 3 by

Diane Gilmour BSc (lions)

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

School of Science University of Tasmania Hobart June, 2006 Chapter 4 In vitro Propagation of Iridaceae

4.1 Introduction

As with the Liliaceae, the majority of work on in vitro propagation of the Iridaceae has been concerned with ornamental flower (and corm) genera. Those that feature prominently in the literature include: , Gladiolus, Freesia and Crocus. Other genera which have been propagated in vitro, but to a lesser extent, include Sparaxis and Schizostylis. Apart from moraea (Sward etal., 1997, 1998), the native Australian members of the Iridaceae have not featured in in vitro propagation literature. However, it is possible that Iridaceae species have been researched and grown in vitro by commercial laboratories who have not published their findings.

4.1.1 In vitro propagation of Iris Iris is a large of approximately 200 (Cooke, 1986; Bryan, 1994) to 300 species (Hitchmough, 1989; Laublin etal., 1991), and more than 2000 (Bryan, 1994). Restricted to temperate and subtropical areas of the northern hemisphere (Cooke, 1986; De Munk and Schipper, 1993; Moore et al., 1993; Bryan, 1994), there are 2 main types: rhizomatous and bulbous (Hessayon, 1995). One species has a corm, and it is sometimes elevated to its own genus (Bryan, 1994). The rhizomatous irises (divided into the bearded and beardless types) are generally larger, evergreen perennials (Cooke, 1986; Moore etal., 1993; Hessayon, 1995), while the bulbous irises (varieties described as Dutch, English and Spanish Iris) are smaller,

328 Chapter 4. In vitro propagation of Iridaceae species with annual and flowers arising from a true bulb (Cooke, 1986; Moore etal., 1993; Hessayon, 1995) (Plate 4.1). Iris flowers are comprised of 3 upright "" known as standards, and 3 drooping ones called the falls (Moore et al., 1993). Rhizomatous types occur in every shade and combination of colour imaginable; while the bulbous types have a more restricted range including: white, cream, yellow, purple and blue (Moore etal., 1993; Hessayon, 1995). Commercial interest in the rhizomatous Iris species is quite low and these are therefore mainly used as ornamental garden (Tompsett, 1985a; De Munk and Schipper, 1993). However, the bulbous species, especially the Dutch Irises are important commercially as cut flowers (Hitchmough, 1989; De Munk and Schipper, 1993), perhaps because they are the only blue flower that is available in large quantities all year round (Tompsett, 1985a). Bulbous irises are also suitable for use in rockeries and borders (Hessayon, 1995) and make an excellent container plant (Hitchmough, 1989; Bryan, 1994).

Plate 4.1. Dutch Iris flowers. Scale bar =2 cm.

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Conventional propagation of the bulbous types is by asexual means. Daughter produced by the original mother bulb are removed and grown on (De Munk and Schipper, 1993). However, this is a slow process, as most species produce less than 5 daughter bulbs annually (Shibli and Ajlouni, 2000). At this rate it takes 10 years to build up commercial quantities of new cultivars (Jehan et al., 1994; Radojevic and Subotic, 1992). Therefore, in vitro propagation methods have been pursued to increase multiplication rates.

The majority of in vitro propagation work on this genus has concentrated on Dutch Irises (De Munk and Schipper, 1993), although rhizomatous irises have also been propagated in vitro (Meyer, 1984; Laublin et al., 1991; Berger et al., 1994; Jehan et al., 1994). The primary explants of choice have included: shoot apices (Baruch and Quak, 1966; Reuther, 1977; Anderson etal., 1990), scale pieces and basal plate sections ( Fujino etal., 1972; Van der Linde etal., 1986, 1988; Van der Linde and Hol, 1988), axillary buds (Fujino et al., 1972), stem sections (Meyer et al., 1975; Hussey, 1976b; Meyer, 1984; Bach, 1988; Yabuya etal., 1991), embryos (Stoltz, 1977; Laublin etal., 1991), small bulblets from the mother bulb basal plate (Allen and Anderson, 1980), (Kromer, 1985), shoots developing naturally on rhizomes (Berger etal., 1994), leaves (Gozu etal., 1993) and bases, flower pieces and apices (Jehan etal., 1994).

In vitro studies of the Iris genus have mainly focussed on increasing the propagation rate, virus eradication, embryo culture and the physiology of flower initiation (De Munk and Schipper, 1993). For shoot regeneration MS medium at full or half strength, with a sucrose concentration between 2-4% has been successful (De Munk and Schipper, 1993). For rhizomatous irises, a modified LS medium has also been used (Meyer, 1984). For optimal regeneration of shoots, NAA (0.03-2.0 mgL -1) was a necessary component of the media (Hussey, 1976b), or equal amounts of NAA and BAP or kinetin (0.5-1.0 mgL -1). Activated charcoal also enhanced shoot regeneration (Bach, 1988). Hussey (1976b) found that a propagation rate of 100 plantlets per original bulb was possible, with a potential rate of up to 1000 or more.

Problems associated with the transfer of plantlets to greenhouse conditions were suggested as being due to bulb size (Mielke and Anderson, 1986). Therefore,

330 Chapter 4. In vitro propagation of Iridaceae species environmental and media factors that would increase bulb size in vitro were studied (lbid). A temperature of 25°C or greater for 6 months followed by continuous darkness with no pre-bulbing chilling period, combined with a 1/2 MS media with 6- 8% sucrose and 0.6% Phytagar was the most successful.

In contrast, to induce bulbing, Van der Linde etal. (1988) interrupted the culture of shoots at 20°C with a 4-week cold treatment at 5°C. The same pre-treatment conditions were also found to increase the number of bulblets produced in the bulblet development stage (Anderson et al., 1990).

Apical meristems have been used as the primary explant type in studies of floral initiation (Doss and Christian, 1979) and virus eradication (Baruch and Quak, 1966; Anderson etal., 1990). The relationship between bulb size, apex size and flowering were studied in the bulbous iris "Ideal". Floral initiation occurred when apical meristems from large, growth-retarded bulbs were cultured on either MS medium containing GA, or on the same medium, without GA, on which scales from large bulbs had been incubated. However, under the same conditions, apical meristems from small bulbs rarely showed initiation. The reduced flowering of the small bulbs was thought to be partly related to characteristics of the apical meristem (Doss and Christian, 1979). For virus eradication, as early as 1966, Baruch and Quak demonstrated that Latent Mosaic Virus could be eliminated by culturing the isolated men stems of Dutch Iris. More recently, a complete micropropagation system beginning with shoot apices and including virus indexing by TEM and ELISA was also developed to produce virus-free Dutch Iris (Anderson etal., 1990). Virus-free bulbous irises have also been obtained by excising and culturing small bulblets that form on the virus-infected mother bulb basal plate (Allen and Anderson, 1980). However, only 12 of the 77 bulblets cultured were found to be free of Iris Mild Mosaic and Iris Severe Mosaic viruses (Ibid.). A method of producing adventitious buds on bulb scale explants has also been used to successfully produce virus-free plants in The Netherlands (Lawson, 1990).

Apart from viruses, another major problem for Iris is that they are very susceptible to bacterial soft rot caused by Erwinia carotovora sub sp. carotovar. Standard procedures for disinfecting shoot explants failed when rhizomes were infected with

331 Chapter 4. In vitro propagation of Iridaceae species bacterial soft rot (Berger etal., 1994). Therefore, a new disinfection protocol was developed. Stock plants weren't irrigated for 2 months prior to the excision of explants; explants were disinfected in ethanol prior to excision from the rhizome and immediately after excision as well as being soaked in 1% (v/v) sodium hypochlorite, followed by mercuric chloride. Using this method, up to 90% of explants taken from infected rhizomes with soft rot symptoms remained free of detectable microorganisms in vitro (Berger et al., 1994).

In an effort to further increase the multiplication rate of irises in vitro, somatic embryogenesis has been studied using commercially important rhizomatous species. Using embryos of I setosa, I pseudacorus and I. versicolor as the primary explant type, following shoot and root growth from the embryo, root and leaf pieces were excised and cultured (Laublin etal., 1991). Clusters of nodular callus formed on root pieces, but not on leaf pieces, on media containing 2,4-D, NAA and kinetin. When callus was transferred to a regeneration medium its surface became covered with white structures resembling young zygotic embryos. Gradually the somatic embryos assumed the typical morphology of monocotyledonous zygotic embryos and quickly germinated. However, they did not form roots unless they were transferred to a medium containing IBA (Laublin etal., 1991). I. setosa, together with I. pumila, has also been propagated through somatic embryogenesis by other researchers (Radojevic etal., 1987; Radojevic and Landre, 1990; Radojevic and Subotic, 1992). A method of rapid multiplication by somatic embryogenesis associated with multibudding has also been developed for I. pallida and I. germanica (Jehan et al., 1994). Callus was obtained from leaf bases, flower pieces or rhizome apices on MS medium, with flower pieces being the most successful. Knudson's orchid agar medium (Knudson, 1946) was used for the embryogenic expression medium. I. germanica has also been propagated by somatic embryogenesis from suspension cell cultures (Shimizu et al., 1997; Wang etal., 1999) and proptoplast cultures (Shimizu et al., 1996). The rare black iris (Iris nigricans) has also been propagated via somatic embryogenesis using callus, cell suspension and protoplast culture systems (Shibli and Ajlouni, 2000).

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4.1.2 In vitro propagation of Gladiolus

The Gladiolus genus has been reported to contain more than 150 species (Hitchmough, 1989; Cohat, 1993; Bryan, 1994), with more than two thirds of these being indigenous to South Africa (Hitchmough, 1989). The other species occur in Eastern and Western Africa, Mediterranean regions, Western Asia and Europe (Hitchmough, 1989; Stanley and Ross, 1989; Cohat, 1993).

Gladiolus plants arise from a corm (Hitchmough, 1989; Stanley and Ross, 1989; Cohat, 1993; Moore etal., 1993; Bryan, 1994; Hessayon, 1995). The annual leaves are sword-like, green and stiff, with overlapping bases (Cohat, 1993; Moore etal., 1993). The inflorescence is a distichous terminal spike (Stanley and Ross, 1989; Cohat, 1993; Moore etal., 1993). Individual flowers are subtended by and are funnel-shaped, with thick waxy petals reaching up to 10 cm across (Stanley and Ross, 1989; Moore etal., 1993) (Plate 4.2). There is a wide range of flower colours available - almost all colours can be found, with the exception of a true blue (Cohat, 1993; Bryan, 1994). Bicolours and tricolours are also available (Moore etal., 1993).

Gladioli are excellent cut flowers (Cohat, 1993; Moore etal., 1993; Bryan, 1994; Hessayon, 1995). In the garden they are best planted in clumps of the same colour in borders (Bryan, 1994), rockeries or banks (Moore et al., 1993). They can also be grown successfully in containers (Bryan, 1994).

Propagation is generally by removal and replanting of new cormlets or by division of the corm into portions containing at least one bud (McKay et al., 1981a). However, some species do not generally produce cormlets, and these must be increased by seed (Hitchmough, 1989). Seed germinates readily, but flowering-sized plants will not be obtained for 2-3 seasons (Hitchmough, 1989).

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Plate 4.2. Gladiolus flower spike. Individual flower diameter = 10 cm.

In vitro propagation has also been used to increase the multiplication of Gladiolus plants. A range of primary explant types have been used to propagate plants in vitro: corm pieces (Hussey, 1975b, 1976; Kumar et al., 1999), leaf pieces (Hussey, 1975b, 1976), ovaries (Hussey, 1975b), scape segments (Ziv et al., 1970; Simonsen and Hildebrandt, 1971; Hussey, 1975b, 1976; Kumar etal., 1999), tissue still enclosed in the spathe (Ziv and Lilien-Kipnis, 2000), root, hypocotyl and leaf explants excised from sterile-grown seedlings (Jager et al., 1998), apical buds from corms (Ziv, 1989), with the most popular being axillary buds from corms (Hussey,

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1977, 1980; Ziv, 1979; Dantu and Bhojwani, 1995). With the exception of leaf and tissue (Hussey, 1975b, 1976) these primary explant sources have all been successful in terms of regeneration capability. Plantlets have been induced to form directly on corm pieces (Hussey, 1975b, 1976; Kumar etal., 1999) and inflorescence stem segments (Ziv etal., 1970; Simonsen and Hildebrandt, 1971; Hussey, 1975b, 1976; Kumar etal., 1999). Callus also formed on corm and inflorescence stem segments, but plantlets could not be regenerated from it (Hussey, 1975b). However, in a later study, shoot differentiation from callus could be obtained on a MS + 1 gM BAP + 1 OgM NAA medium, or by omitting the PGRs and subjecting plants to a heat shock (50°C for 1 hour) (Kumar et al., 1999).

Dormancy has been a problem encountered in Gladiolus cultures. When grown on a basal medium (without PGRs) Gladiolus plantlets grew for 6-10 weeks and then became dormant. However, dormancy could be prevented by culturing on 0.03 mgL -1 BAP, which also helped to increase multiplication by promoting branching (Hussey, 1976a). As well as preventing dormancy, BAP also promoted shoot growth and inhibited root development (Hussey, 1977). It was more successful to subculture plantlets to media with a lower BAP concentration - they formed resting-corms which were not dormant when transplanted (Hussey, 1977). A pre-transplanting medium containing a 1/2 strength MS + 0.3% AC and NAA, was also successful in preventing post-transplant dormancy (Ziv, 1979). While, more recently, in vitro- formed corms showed 100% germination in the field following a cold treatment of 5°C for 8 weeks (Dantu and Bhojwani, 1995).

Gladiolus cultures have been widely grown on a solid MS medium or a slightly modified MS (Hussey, 1975b, 1976, 1980; Ziv, 1979; Jager etal., 1998; Kumar et al., 1999). A 1/2 strength MS (solid) has also been used (Hussey, 1977; Ziv, 1989). Liquid MS (full or 1/2 strength) has been used in an effort to decrease the extensive manual handling required in a micropropagation system, and to increase multiplication rates (Ziv, 1989; Steinitz etal., 1991; Dantu and Bhojwani, 1995). Some problems do occur in liquid cultures, such as the development of abnormal leaves. Therefore, Ziv (1989) used growth retardants to prevent leaf development. In the presence of growth retardants, bud explants proliferated into massive bud

335 Chapter 4. In vitro propagation of Iridaceae species aggregates without leaves. The buds developed into protocorms which formed comilets following subculture to solid medium. These cormlets weren't dormant and after transplanting developed into plantlets (Ziv, 1989).

A high concentration of sucrose in the culture medium has also produced favourable results in Gladiolus cultures (Steinitz et al., 1991; Kumar et al., 1999). Gladiolus shoot cultures could be induced to regenerate corms early and rapidly in liquid shake cultures in a medium containing a high concentration of sugar, together with an adequate level of cytokinin and a GA biosynthesis inhibitor (Steinitz et al., 1991). Also, when combined with a heat shock, shoot cultures on a high sucrose medium rooted prolifically (Kumar etal., 1999). They also showed a better survival (90%) when transplanted to ex vitro conditions than those on media with a normal sucrose concentration (40% survival) (Kumar et al., 1999).

More recently, in vitro studies of Gladiolus have focused on more advanced techniques such as the genetic transformation of plants, with the aim being to confer resistance to viral and fungal pathogens that decrease the production of Gladiolus plants and flowers. The stable transformation of Gladiolus plants has been achieved using suspension cells and callus (Kamo etal., 1995). Suspension cells and callus were subjected to particle-bombardment with PAT- and GUS-expressing plasmids. Stably-transformed calli were screened for on selection media and then transferred to regeneration media to recover transgenic plants. More than 100 transgenic plants were recovered (Ibid.).

Possible selective agents and marker genes for use in genetic transformation of Gladiolus have also been studied (Chauvin et al., 1996, 1997). The in vitro behaviour of cormel slices was analysed in the presence of the most commonly used selective agents: kanamycin, hygromicin and phosphinotricin (Basta ®). Basta ® proved to be the most effective agent for Gladiolus. It inhibited all regeneration even at a low concentration in the media. Therefore, the transferred DNA to be used must contain resistance genes to phosphinotricin. GUS activity was not detected in any explants of Gladiolus, meaning that this marker gene can be used in the preliminary steps of transformation procedures (Chauvin etal., 1996, 1997). Kanamycin was initially thought to be an unsuccessful selective agent (Chauvin etal., 1997).

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However, being unable to use this agent meant that the npt II gene could also not be used. Being unable to use this gene is a hindrance in the genetic engineering of this species because most of the molecular constructs available utilise it (Chauvin et al., 1999). Therefore, the regeneration of cormel slices was investigated on media solidified with different gelling agents and with various concentrations of kanamycin (Ibid.). Regeneration was observed on media containing a high concentration of kanamycin with one of the gelling agents (a K-carrageenan), but with others, 50% of this concentration was generally sufficient to totally inhibit regeneration. Therefore transformation using kanamycin as a selective agent is possible for Gladiolus (Chauvin et al., 1999).

In vitro selection for resistance to Fusarium oxysporum f. sp. gladioli, one of the main pathogens of Gladiolus has also been attempted (Remotti et al., 1996). Using cell suspension cultures the potential of fusaric acid as a selective agent was evaluated. This chemical was chosen as the Fusarium fungus produces it both in vitro and in vivo, and a correlation was found between the sensitivity of a number of Gladiolus genotypes for fusaric acid and their resistance to the fungus (Ibid.). The majority of cells from a cell suspension culture were killed by 0.12mM fusaric acid, but a number of callus clumps which emerged from surviving cells had a lower sensitivity for the chemical. Growth of the fungus on the selected callus was retarded at least 50% compared to the control (Ibid.). This research has huge potential not only for Gladiolus, but a wide range of other ornamental species.

4.1.3 In vitro propagation of Freesia The Freesia genus contains 11 species (Stanley and Ross, 1989; Imanishi, 1993; Bryan, 1994), which are all native to South Africa (Hitchmough, 1989; Stanley and Ross, 1989; Imanishi, 1993; Bryan, 1994). Only 1 species, F. lactea (syn. F. refracta var. alba) is in general cultivation (Hitchrnough, 1989). The large flowered freesias grown for cut flowers are hybrids of species such as F. refracta, and are referred to as F. x hybrida (Hitchmough, 1989).

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Freesias grow from a tunicated corm (Stanley and Ross, 1989; Imanishi, 1993; Hessayon, 1995). The annual leaves are broadly linear, conspicuously ribbed in most species (Hitchmough, 1989) and are mostly basal (Stanley and Ross, 1989). The inflorescence is a one-sided spike (Hitchmough, 1989; Stanley and Ross, 1989; Imanishi, 1993; Bryan, 1994), bearing the usually heavily scented, zygomorphic, campanulate flowers (Hitchmough, 1989; Bryan, 1994) in an upward facing direction (Imanishi, 1993; Bryan, 1994). There is a wide range of flower colours available: whites, creams, yellows, oranges, pinks, reds, blues and violets (Hitchmough, 1989; Bryan, 1994; Hessayon, 1995).

Freesias are widely used as, and make excellent, cut flowers (Smith and Danks, 1985; Bryan, 1994). They also grow well in containers (Bryan, 1994). As garden plants, they are only suited to Mediterranean type climates as they are not very hardy and need a long growing season at a cool temperature (Imanishi, 1993). Propagation is usually by removal of cormlets from the mother corm, by seed (Smith and Danks, 1985) or by micropropagation (Imanishi, 1993).

A number of primary explant types have been used to try to initiate cultures of Freesia: corm pieces (Hussey, 1975a,b, 1976), leaves (Hussey, 1975a,b), ovary tissue (Hussey, 1975b, Bach, 1987), sections of inflorescence stem (Hussey, 1975a,b; Wang etal., 1994), leaves on young inflorescence stems (Wang et al., 1993, cited in: Zaidi etal., 2000), excised flower buds (Pierik and Steegmans, 1975, 1976), etiolated sprouts (Doi etal., 1992) and immature zygotic embryos (Wang et al., 1993, cited in: Zaidi etal., 2000). The majority of explant types mentioned above successfully regenerated. However, leaves could not be induced to regenerate plantlets or callus (Hussey, 1975a,b). Ovary tissue also did not show any regeneration in these studies (Hussey, 1975a,b). However, young ovaries were successfully used as primary explants in a study comparing the regeneration of 20 Freesia cultivars in a later study (Bach, 1987). Plantlets were not induced directly on inflorescence stem tissue (Hussey, 1975b) but callus was, and it was possible to regenerate plantlets from this callus. Plantlets have also been regenerated from callus in another study (Bajaj and Pierik, 1974). Callus is not usually recommended in a micropropagation system due to the often associated somaclonal variation (Dodds and Roberts, 1985; Stewart,

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1999b). However, in the case of Freesia the callus is genetically stable (Van Aartrijk and Van der Linde, 1986), so a method of utilising proliferation of callus with subsequent regeneration of plantlets is suitable.

Freesia responds well to in vitro conditions. In broad studies comparing regeneration in vitro of a number of Iridaceae genera (Freesia, Gladiolus, Iris, Sparaxis and Schizostylis), Freesia was found to be the most consistently reactive, with abundant plantlets being regenerated from corm tissue or callus (Hussey, 1975b, 1976). Dormancy occurred in all Iridaceae species after plantlets were cultured for a certain time length (12-14 weeks for Freesia) (Hussey, 1976a), but it could be broken in all species by the addition of cytokinin. Freesia, however, did not require cytokinin to prevent dormancy - growth resumed if crowns were cut out, split in half and placed on new basal media (Hussey, 1976a).

In more recent work, somatic embryogenesis has been studied in Freesia refracta (Wang et al., 1994). Young inflorescence stems were used as primary explants. Using a media supplemented with IAA (2 mgL -1) and BAP (3 mgL-1) it was possible to directly initiate somatic embryos on the inflorescence stem tissue (Wang et al., 1994). Subsequent regeneration of plants from these embryos was not discussed.

The use of CO2-enriched atmospheric conditions in the rooting stage was studied by Doi et al. (1992). Root systems developed well and shoots grew vigorously under these conditions. Plantlets also acclimatised more easily to ex vitro conditions than those cultured under ambient atmospheric conditions (Doi et al., 1992).

Freesia hybrids (Hussey, 1975b, 1976) and cultivars (Pierik and Steegmans, 1976; Bach, 1987) have been the most widely used subjects for in vitro studies. Freesia refracta has also been used (Wang et al., 1993, 1994). A solid medium is generally used for in vitro propagation of Freesia, usually a slight modification of MS (Hussey, 1975b, 1976, 1980; Pierik and Steegmans, 1976; Doi et al., 1992). However, a liquid MS has also been used (Doi et al., 1992). For initiation of somatic embryos, N-6 medium was used (Wang et al., 1994).

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4.1.4 In vitro propagation of Crocus There are 80 species in the Crocus genus (Hitchmough, 1989; Moore etal., 1993; Bryan, 1994). The natural distribution is entirely confined to the northern hemisphere (Hitchmough, 1989), occurring in Mediterranean regions, the Alps, Pyrenees, Turkey and Iran (Bryan, 1994). Although the majority of species flower in autumn and winter (Bryan, 1994), it is the spring flowering cultivars and species that are the most important horticulturally (Benschop, 1993). C. sativus, the "saffron crocus", is the main representative of the autumn flowering group (Benschop, 1993). The spice saffron is obtained from the dried stigmas of this species (Fakhrai and Evans, 1989, 1990; Sarma etal., 1990; Benschop, 1993). Saffron has a wide range of uses, mainly as an additive to colour and flavour food (Falchrai and Evans, 1990), but also as a textile dye (Basker and Negbi, 1983; Moore et al., 1993), in drugs, medicine and perfumes (Ingram, 1969; Basker and Negbi, 1983).

Crocus has a tunicated corm (Moore et al., 1993; Hessayon, 1995). The leaves are grass-like, with rolled edges which expose a white or silver streak along the midrib (Bryan, 1994; Hessayon, 1995). The goblet-shaped flowers have 6 petals (Hessayon, 1995) and are held upright on short scapes to 10 cm long (Moore etal., 1993) (Plate 4.3). They are available in a wide range of colours: whites, creams, yellows, oranges, mauves, purples and blues (Moore etal., 1993; Bryan, 1994; Hessayon, 1995), many are striped (Bryan, 1994) and have conspicuous feathery stigmas (Moore etal., 1993) (Plate 4.3).

Although Crocuses are small, they are very versatile horticulturally, being useful in rockeries (Hessayon, 1995), in beds or borders (Bryan, 1994; Hessayon, 1995), as container plants and for naturalising in lawns (Moore etal., 1993; Bryan, 1994; Hessayon, 1995).

The majority of studies on in vitro propagation of the Crocus genus have used the commercially important Crocus sativus. Initially, work centred on increasing the naturally slow reproductive rate of corm formation by using in vitro techniques (Ding etal., 1979, 1981; Homes etal., 1987; Ilahi et al., 1987; Isa and Ogasawara, 1988). However, by the late 1980s the focus had switched to the possible production

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of saffron pigments in vitro, as an alternative to the use of whole plants, and to help meet the growing demand for the spice (Himeno and Sano, 1987; Sano and Himeno, 1987; Honi etal., 1988; Faldrai and Evans, 1990; Sartna etal., 1990).

Plate 4.3. Crocus plant. Note the conspicuous feathery stigmas. Flower diameter = 5 cm.

A wide range of primary explant types have been used to initiate cultures of Crocus sativus. For experiments aimed at increasing the natural reproductive rate, corms or corm pieces were the most popular explant source (Ding et al., 1979, 1981; Homes et al., 1987; Isa and Ogasawara, 1988; Milyaeva etal., 1995). Direct regeneration of minicorms (Homes etal., 1987; Milyaeva etal., 1995) or buds (Ding etal., 1979) from the surface of the corm explants occurred. In other cases, an intermediate callus stage occurred (Ding et al., 1981; Ilahi et al., 1987; Isa and Ogasawara, Milyaeva et al., 1995) with subsequent regeneration of shoots (Ding et al., 1981; Ilahi et al., 1987; Isa and Ogasawara, 1988). Minicorms also developed from embryoid structures on explant calluses (Milyaeva etal., 1995). Somatic embryogenesis has also been achieved in Crocus sativus (George etal., 1992; Ahuja etal., 1994) and Crocus cancellatus (Karamian and Ebrahimzadeh, 2001).

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For in vitro production of saffron, primary explants of a floral origin have been used: ovaries and/or half-ovaries (Chichiricco and Grilli Caiola, 1987; Himeno and Sano, 1987; Sano and Himeno, 1987; Fakhrai and Evans, 1990), stigmas (Sano and Himeno, 1987; Falchrai and Evans, 1990; Sarma etal., 1990), pistils (Hon et al., 1988), anthers and petals (Fakhrai and Evans, 1990). -like structures regenerated either indirectly through an intermediate callus stage (Himeno and Sano, 1987; Sano and Himeno, 1987; Falchrai and Evans, 1990) or directly (Falchrai and Evans, 1990; Sarma etal., 1990) on all explants listed above. Falchrai and Evans (1990) utilised a range of explant types, and found that half-ovaries performed the best, producing up to 25 stigma-like structures per explant. Sarma et al. (1990) used only stigma explants, but at various stages of development. Best results occurred when stigmas were at a yellowish-orange stage, with up to 18 stigma-like structures regenerated per explant. The addition of NAA to the media was found to be crucial for the development of stigma-like structures (Fakhrai and Evans, 1990; Sarma et al., 1990). Analysis of the pigments produced by in vitro stigmas by thin layer chromatography (Falchrai and Evans, 1990) and HPLC (Sarma et al., 1990) showed that they were very similar to those from naturally-produced stigmas. However, safranal was not detected in fresh samples (Sarma et al., 1990). Himeno and Sono (1987) as well as Sano and Himeno (1987) also did not detect safranal. Crocin and picrocrocin pigments were detected, and after a heat treatment, safranal did appear.

Crocus cluysanthus was the only spring-flowering Crocus to have been propagated in vitro until recently (Benschop, 1993). A wide range of explant types of this species (roots, leaves, corm pieces, basal plates, petals, ovaries and anthers) were cultured on a basal MS medium with 20 different combinations of either kinetin and NAA or BAP and 2,4-D (Fakhrai and Evans, 1989). Only ovary explants on media containing more than 5mgL-1 BAP showed any regeneration. Callus and subsequently stigma-like structures were formed. Callus could also be induced to form corms and shoots with the addition of 0.5mgL -1 2,4-D to the medium.

More recently, Choob etal. (1994) did a comparison of the regenerative capabilities of 4 spring-flowering Crocus species in vitro. Using , , styles with stigmas, ovaries and ovules as explants, C. sieberi, C. vernus, C. chrysanthus and C.

342 Chapter 4. In vitro propagation of Iridaceae species aureus, were compared. C. sieberi and C. vernus were found to have low capacities for both callusogenesis and morphogenesis, compared to C. chrysanthus and C. aureus, which rapidly produced callus and regenerated organs. Except for anthers and ovules, all explants formed callus that was capable of regeneration. Ovary wall explants, and to a lesser degree explants, had the highest morphogenic capacity. Stigma and style-like organs were usually regenerated, while in some cases, shoots developed (Choob etal., 1994).

A solid MS has been the basal medium most widely used for in vitro propagation of Crocus (Ding et al., 1979, 1981; Chichiricco and Grilli Caiola, 1987; Homes et al., 1987; Ilahi etal., 1987; Isa and Ogasawara, 1988; Falchrai and Evans, 1989; George etal., 1992; Milyaeva etal., 1995). A semi-solid MS has also been used (Sarma et al., 1990). As well as MS, Homes etal. (1987) used B5 (Gamborg etal., 1968) and combinations of the two. Whites' basal medium (1963) was used by Falchrai and Evans (1990), while Sano and Himeno (1987) used LS (Linsmaier and Skoog, 1965) and Nitsch (1972) media.

4.1.5 Aims of the current study The aim of the present study was to utilise the known information for in vitro propagation of other Iridaceae species to develop complete micropropagation procedures for the species included in this study, the majority of which have never been propagated using these techniques before.

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4.2 Materials and Methods

4.2.1 In vitro propagation of

4.2.1.1 Explant source: Seeds

4.2.1.1.1 Disinfestation Seeds were disinfested following the procedure outlined in section 2.2.3.1.2. Seeds were immersed in 2% Na0C1 for 20 min. Following disinfestation and rinsing, individual seeds were placed in 30 mL culture tubes containing approximately 10 mL of basal MS medium. They were incubated in the light under standard culture conditions.

4.2.1.1.2 Multiplication Seedlings (3.5 months old) were subcultured (usually cut in half, retaining roots on each half where possible) and placed into larger 100 i-nL tubes containing approximately 25 mL of the multiplication media listed in Table 4.1 (20 seedlings in each treatment).

Table 4.1. Multiplication media treatments for Diplarrena moraea seedlings.

Multiplication media treatments

1. MS (Control) 4. MS + 21AM 2iP

2. MS + 21.1M BAP 5. MS + 1281.1.M 2iP

3. MS + 21AM kinetin 6. MS + 124M BAP

After one month on the new media seedlings were assessed for multiplication by counting the number of new shoots produced. Following the first subculture (3 months after seedlings were placed on the multiplication media), the number of shoots obtained were again counted and recorded. From this information the mean number of new shoots produced by shoots growing on each multiplication media treatment was determined. This data was analysed by ANOVA and Tukey's HSD tests. A proportion of shoots on each multiplication media treatment had died by the

344 Chapter 4. In vitro propagation of Iridaceae species time shoots were subcultured. This data was analysed using Pearson's Chi-Squared statistic.

4.2.1.1.3 Rooting Shoots from the multiplication media experiment also grew roots on some of the treatments (control, MS + ZAM BAP, MS + ZAM kinetin and MS + 20VI 2iP). Therefore, rather than doing a separate rooting media experiment, some of the plantlets from these treatments were transplanted to ex vitro conditions.

4.2.1.1.4 Transplanting One hundred and fifty D. moraea rooted plantlets were transplanted to ex vitro conditions. They were removed from culture tubes very carefully, media was washed off the roots in lukewarm water and they were planted in pasteurised potting mix in seedling trays. The mix was made up of 1 part sand: 1 part peat: 1 part perlite. This mix was used successfully for ex vitro establishment of B. grandiflora plantlets (Johnson, 1996a) and the Liliaceae species included in this study (see Chapter 3).

Plantlets were grown in a greenhouse under mist, with the intensity of the natural light reduced to 70% using shadecloth. They were sprayed with fungicide (Previcur, Shering Pty Ltd Agrochemicals) to avoid fungal contamination. Four weeks after planting, a half-strength Aquasol liquid fertiliser was applied twice weekly. Plantlets were checked one month after planting out, and any dead plants were counted and removed. This process was repeated 2 months later.

4.2.2 In vitro propagation of Diplarrena latifolia

4.2.2.1 Explant source: Seeds In vitro germinated D. latifolia seedlings obtained from the "D. latifolia in vitro media type and kinetin experiment" (Chapter 2, section 2.2.3.2.1) were used for this experiment.

345 Chapter 4. In vitro propagation of Iridaceae species

4.2.2.1.1 Disinfestation Seeds were disinfested following the procedure outlined in section 2.2.3.2.1. Seeds were immersed in 2% Na0C1 for 20 min. Following disinfestation and rinsing, individual seeds were placed in 30 mL culture tubes containing approximately 10 mL of media. Seven different media treatments were used: MS (control), EC, and MS supplemented with 0.51.IM, 21.1M, 81.LM, 321.1M and 128gM kinetin. For the current experiment, seedlings that had germinated on MS, EC and MS + ZAM kinetin were used for multiplication media experiments.

4.2.2.1.2 Multiplication Seedlings were initially cut in half and placed on MS + 2A4 BAP to multiply until enough shoots were available for a larger scale multiplication media experiment. Following this initial multiplication phase the shoots were transferred to MS + 1 gl.:1 AC for 4 weeks to remove the PGRs from the media. The shoots were then transferred to the multiplication media to be tested (Table 4.2). The very high cytokinin concentration (128A4) included in the multiplication media experiment for D. moraea was not used for multiplication of D. latifolia due to the callus development that occurred (on this concentration) in the experiment with the former species.

Table 4.2. The number of D. latifolia shoots placed on each multiplication media treatment. Reps = Replicates.

Media treatment Number of shoots 1. MS (control) 30 (6 reps of 5) 2. MS + 21.tM BAP 30 (6 reps of 5) 3. MS + 21.tM 2iP 30 (6 reps of 5) 4. MS + 2gM Kinetin 30 (6 reps of 5) 5. MS + 81.t.M BAP 30 (6 reps of 5) 6. MS + 81../M 2iP 30 (6 reps of 5) 7. MS + 81A.M Kinetin 30 (6 reps of 5) 8. MS + 321AM BAP 30 (6 reps of 5) 9. MS + 321.1M 2iP 30 (6 reps of 5) 10. MS + 321AM Kinetin 30 (6 reps of 5) TOTAL 300

346 Chapter 4. In vitro propagation of Iridaceae species

After one month on the new media seedlings were assessed for multiplication by counting the number of new shoots produced. Following the first subculture (approximately 3 months after seedlings were placed on the multiplication media), the number of shoots obtained were again counted and recorded. From this information the mean number of shoots produced by seedlings growing on each multiplication media treatment was determined. This data was analysed by ANOVA and Tukey's HSD tests.

4.2.2.1.3 Rooting Shoots from the multiplication media experiment were used for this experiment. The majority of shoots growing on the control, MS + ZIM BAP, MS + 21.LM 2iP and MS + 211M kinetin treatments, and a small proportion of those growing on the MS + 811M concentrations (of all cytokinins), grew roots as well as shoots on these treatments. Therefore, due to the success achieved with transplanting D. moraea directly from the multiplication stage, these D. latifolia rooted plantlets were also transplanted directly from multiplication media. The shoots growing on other treatments that did not grow roots were transferred to MS media, as it was hoped that by reducing the level of cytokinin in the media to zero, root growth may be stimulated.

4.2.2.1.4 Transplanting Two hundred of the D. latifolia rooted plantlets were transplanted to ex vitro conditions directly from the multiplication media. The transplanting procedure, potting medium and post-transplanting conditions were as described previously for D. moraea (section 4.2.1.1.4). Plantlets were checked one month after planting out, and any dead plants were counted and removed. This process was repeated 2 months later.

347 Chapter 4. In vitro propagation of Iridaceae species

4.2.2.2 Explant source: Whole immature flower buds

4.2.2.2.1 Disinfestation Floral scapes with unopened flower buds were collected from a population of D. latifolia at Mt Field, Tasmania in late October. The buds were divided into 3 different stages of development, and as such labelled 1-3, with 1 being the oldest, most mature buds, 3 being the youngest, most immature buds, and stage 2 being intermediate between the two. Upon return to the laboratory the floral scapes were stored at 4°C until disinfestation the following day.

For disinfestation, the intact scapes were placed in long polycarbonate tubes (500 mL capacity), and the tops of the tubes were covered with gauze (attached with elastic bands). Tubes containing scapes were placed under running tap water for 1 hr. The water was then drained out, a 2% Na0C1+ 0.1% Tween 20 solution was added and the sealed containers were agitated on a rotary shaker for 30 min. This solution was poured out under aseptic conditions and the scapes were rinsed twice in SDW. Following disinfestation, individual flower buds + a small length (— 1 cm) of the were removed from the scape, rinsed again in SDW, and placed in 100 mL culture tubes containing approximately 30 mL of media. The pedicels were gently pushed into the media surface. The explants were placed in the media used successfully for whole flower buds of B. grandiflora by Bunn and Dixon (1997), referred to as FBM (1/2 MS + 101AM BAP + 0.51AM IBA) (Table 4.3). They were checked frequently for growth or any changes, and the number and percentage of explants that had grown shoots was recorded at bi-monthly intervals. Results were not analysed statistically due to low sample numbers.

Table 4.3. The number of D. latifolia flower buds from each developmental stage placed on FBM.

Developmental stage No. of buds on FBM 1 8 2 8 3 7 Total 23

348 Chapter 4. In vitro propagation of Iridaceae species

4.2.2.2.2 Multiplication After approximately 5.5 months on the initiation media shoots that had developed on Stage 1 and 2 flower buds were removed and placed on the multiplication media (FBMM). This was the same media used for the multiplication of B. grandiflora shoots that had originated from flower bud explants (Bunn and Dixon, 1997), and the Liliaceae species studied in the previous chapter. The shoots were subcultured after 4.5 months on the multiplication media, and at this time the number of new shoots produced by each original shoot was counted and recorded. From this information, the mean number of shoots produced by shoots (originally from flower buds at each developmental stage) was calculated. Unfortunately results could not be analysed statistically due to such a small number of shoots being produced from the flower bud explants.

4.2.3 In vitro propagation of Isophysis tasmanica

4.2.3.1 Explant source: Seeds Seeds were used as the initial explant type. They were chosen as the rhizomes of I. tasmanica are very short, and it was impossible to find any pieces with buds. No young shoots were developing either, so these could not be used as an explant source.

4.2.3.1.1 Disinfestation Seeds were disinfested in 2% Na0C1 for 20 min following the procedure outlined in Chapter 2 (section 2.2.3.3.1). After disinfestation and rinsing they were placed in 30 mL tubes containing approximately 10 mL of MS media (1 seed per tube). They were incubated in the dark at 25°C. Dark conditions were chosen as seeds germinated best in the dark in previous seed germination experiments (See Section 2.3.2.3.1).

4.2.3.1.2 Multiplication

4.2.3.1.2.1 Initial multiplication media experiment (fbr increasing shoot numbers)

Five months after placement on MS media, the seedlings were large enough to be used for further experimentation. They were placed onto 3 different multiplication

349 Chapter 4. In vitro propagation of Iridaceae species media, all containing the same concentration of three different cytolcinins (Table 4.4), with the aim being to increase shoot numbers so that a larger experiment using more multiplication media treatments could be performed.

Table 4.4. The number of Isophysis tasmanica seedlings placed on each initial multiplication media treatment.

Media treatment Number of seedlings 1. MS + 2i_tM kinetin 27 (in 5 tubes) 2. MS + 211M BAP 29 (in 13 tubes) 3. MS + 21.LM 2iP 20 (in 4 tubes)

After seedlings had been growing on the multiplication media for 5.5 months, they were assessed in terms of the percentage of seedlings that produced new shoots, the percentage of seedlings that didn't produce any new shoots, and the total and mean number of shoots produced by seedlings grown on each media treatment.

Following the assessments, shoots were subcultured and all were placed onto MS + 2IAM BAP media to continue increasing shoot numbers for further experimentation. This media was chosen as, although the highest mean number of new shoots occurred on the MS + ZAM 2iP treatment, shoots appeared healthier on MS + 21.1M BAP.

4.2.3.1.2.2 Multiplication media experiment

After shoot numbers had increased enough to perform the multiplication media experiment, shoots were transferred to a MS medium supplemented with 1 g1: 1 AC. Shoots remained on the MS + AC media for approximately 1 month before they were transferred under aseptic conditions to the 16 multiplication media to be tested (Table 4.5). The mean number of shoots data for each treatment was analysed by ANOVA and Tukey's HSD tests.

350 Chapter 4. In vitro propagation of Iridaceae species

• Table 4.5. Multiplication media for Isophysis tasmanica shoots, and the number of shoots grown on each treatment.

Multiplication media treatments Number of shoots 1.MS (control) 30 (6 replicates of 5 shoots) 2. MS + 0.51AM kinetin 30 (6 replicates of 5 shoots) 3. MS + 21.iM kinetin 30 (6 replicates of 5 shoots) 4. MS + 81.IM kinetin 30 (6 replicates of 5 shoots) 5. MS + 321AM kinetin 30 (6 replicates of 5 shoots) 6. MS + 128W kinetin 30 (6 replicates of 5 shoots) 7. MS + 0.5p.M 2iP 30 (6 replicates of 5 shoots) 8. MS + 21.IM 2iP 30 (6 replicates of 5 shoots) 9. MS + 81AM 2iP 30 (6 replicates of 5 shoots) 10.MS + 32p.M 2iP 30 (6 replicates of 5 shoots) 11. MS + 124/M 2iP 30 (6 replicates of 5 shoots) 12. MS + 0.51.1M BAP 30 (6 replicates of 5 shoots) 13. MS + 2p,M BAP 30 (6 replicates of 5 shoots) 14. MS + 8i.i.M BAP 30 (6 replicates of 5 shoots) 15. MS + 321.LM BAP 30 (6 replicates of 5 shoots) 16. MS + 12811M BAP 30 (6 replicates of 5 shoots)

4.2.3.1.3 Rooting Shoots from the multiplication media experiment were used for this experiment. Five hundred shoots were grown on a MS + 1 gL -1 AC media for 6 weeks. They were then going to be grown on a number of different rooting media treatments.

Shoots from all initial treatments grew roots on the AC media, and therefore a rooting media experiment was not pursued. Instead, these rooted plantlets were transplanted to ex vitro conditions after 8 weeks on the AC media.

4.2.3.1.4 Transplanting Four hundred rooted I. tasmanica plantlets were transplanted to ex vitro conditions. The transplanting procedure, potting medium and post-transplanting conditions were all the same as previously described for D. moraea (section 4.2.1.1.4). A total of 162 plantlets were transplanted. They were checked one month after planting out, and any dead plants were counted and removed. This process was repeated 2 months

351 Chapter 4. In vitro propagation of Iridaceae species later. The remaining plants were repotted approximately 6 months after transplanting and 12 months later the survival of plants was determined (as a percentage of the original number of plants transplanted from tissue culture).

4.2.3.2 Explant source: Whole immature flower buds

4.2.3.2.1 Disinfestation Eleven floral scapes, each with an unopened (immature) flower bud, were collected from a population of I. tasmanica at "The Needles", SW Tasmania in late December. Upon return to the laboratory the floral scapes were stored in sealed plastic bags at 4°C until disinfestation 3 days later.

The disinfestation procedure was the same as that used for D. latifolia flower buds (section 4.2.2.2.1). The explants were placed on FBM, the media used successfully for whole flower buds of B. grandiflora by Bunn and Dixon (1997) and the Liliaceae species studied in the previous chapter. They were checked frequently for growth or any changes.

352 Chapter 4. In vitro propagation of Iridaceae species

4.3 Results

4.3.1 In vitro propagation of Diplarrena moraea

4.3.1.1 Explant source: Seeds

4.3.1.1.1 Disinfestation The disinfestation procedure was successful with 100% of seeds remaining contaminant free. Seeds began to germinate within 10 days and 100% germination was achieved within 60 days.

4.3.1.1.2 Multiplication One month after placement on the multiplication media, the mean number of shoots produced by seedlings had increased when the culture medium was supplemented with a cytokinin (Fig. 4.1) (Plate 4.4). The greatest proliferation occurred in the treatment containing MS + 128pM 2iP, which showed a 7-fold increase over that of the control.

1 0

7.5

Mean number of new shoots

2.5

MS MS+2AM MS+1281.1M MS+211M MS+ZAM MS+1281AM BAP BAP kinetin 21P 2iP

Treatment

Figure 4.1. The effect of different concentrations of the cytokinins BAP, kinetin and 2iP on the mean number of new shoots produced by D. moraea subcultured seedlings after 1 month on multiplication media.

353 Chapter 4. In vitro propagation of lridaceae species

Plate 4.4. Diplarrena moraea seedlings on multiplication media. Left to right: Control (MS), MS + 2uM 2iP, MS + 21.iM kinetin, MS + 21.tM BAP, MS + 128uM 2iP, MS + 128[IM BAP. Tube diameter = 2.5 cm.

Following the first subculture, the mean number of new shoots produced by seedlings had increased in each treatment, but the pattern of the graph had not changed from the results obtained one month after placement on the multiplication media (Fig. 4.2).

30

20 Mean number of new shoots 10

1

MS MS+21.LM MS+12131.1M MS+2ptki MS+ZAM MS+1281.1M BAP BAP kinetin 2iP 2iP

Treatment

Figure 4.2. The effect of different concentrations of the cytokinins BAP, kinetin and 2iP on the mean number of new shoots produced by D. moraea subcultured seedlings after 3 months on multiplication media.

354 Chapter 4. In vitro propagation of Iridaceae species

The ANOVA showed that there were differences between multiplication media treatment means (p = 0.000, Table 4.6). As expected the mean number of new shoots increased with increasing cytokinin concentration for BAP and 2iP (Fig. 4.2). For both of these cytokinins, the highest concentration (128W) produced significantly more shoots than the control (Table 4.7); while for 2iP the highest concentration produced significantly more shoots than the lowest concentration (2).tM) (Table 4.7). However, although very high numbers of new shoots were produced by seedlings at the highest cytokinin concentration (for both BAP and 2iP) (Plate 4.5), the majority of these seedlings had also developed callus. At the lower 2i1M concentration, seedlings on the media containing BAP produced the most new shoots (Fig. 4.2). However, this result was not significantly greater than those for 2iP or kinetin at the same concentration (Table 4.7). The 21AM concentrations of all cytokinins also did not produce significantly higher mean shoot numbers than the control (Table 4.7). Seedlings growing on the control treatment and the 21A4 concentrations of all cytokinins tested also grew roots (Plate 4.6).

Table 4.6. One-Way Analysis of Variance for mean number of shoots produced by D. moraea seedlings on multiplication media.

Source Sum of Squares Df Mean Square F-Ratio P-value A: Between Treatments 3673.677 5 743.735 10.335 0.000 B: Within Treatments 3056.853 43 71.090 TOTAL 6730.531 48

Table 4.7. Tukey's HSD test groupings of D. moraea multiplication media treatments (Method: 95.0 percent HSD). Values followed by the same letter are not significantly different (p = 0.05).

Treatment Mean 1. MS (Control) 3.67 a 2. MS + 21.tM BAP 7.11 ab 3. MS + 12804 BAP 16.57 bc 4. MS + 21AM kinetin 4.88 ab 5. MS + 211M 2iP 3.75a 6. MS + 128uM 2iP 27.38 c

355 Chapter 4. In vitro propagation of Iridaceae species

A proportion of shoots from all treatments were dead at the time seedlings were subcultured (Fig. 4.3). The proportion of dead shoots was similar for all treatments, ranging from 21.6% (MS + 1281.1M BAP) to 35.9% (MS + 21.LM kinetin). Pearson's Chi-squared statistic showed that there were no significant differences between treatments in regard to the proportion of dead shoots (x2 = 0.5; p = 0.99).

100

75 —

Percentage 50 _ of dead shoots 25 - 1 MS MS+4IM MS+21.1M MS+21.LM MS+128 MS+128gM 2iP kinetin BAP 2iP BAP Treatment

Figure 4.3. The percentage of D. moraea shoots that had died by the time of the first subculture of shoots growing on various multiplication media treatments.

356 Chapter 4. In vitro propagation of Iridaceae species

Plate 4.5. Diplarrena moraea subcultured shoots on multiplication media: MS + 128p.M BAP (a) and MS + 1281.1M 2iP (b). Note the very high number of shoots compared to those in Plate 4.6 and the lack of any root development. Also note the presence of some dead leaves in both tubes. Tube diameters = 2.5 cm.

Plate 4.6. Diplarrena moraea subcultured shoots on multiplication media: MS (a) and MS + 211M kinetin (b). Note the lack of multiplication on MS, and the low level of multiplication on MS + 21.1M kinetin. Also, note the development of roots on both media. The strong root development has actually pushed the shoot out of the media (a). Scale bars = 0.5 cm.

357 Chapter 4. In vitro propagation of Iridaceae species

4.3.1.1.3 Rooting All of the shoots growing on the following multiplication media treatments grew roots as well as shoots: MS (control), MS + 21.iM BAP, MS + 21iM 2iP and MS + 21iM kinetin (Plate 4.6). Therefore, a separate rooting media was not required. Roots began to grow 4 weeks after shoots were placed on the multiplication media.

4.3.1.1.4 Transplanting Transplanting was initially quite successful, with 100% of plantlets still alive 1 month after their removal from tissue culture conditions. However, 2 months later the proportion of surviving plants had decreased to 63.3%. The majority that died had rotted, apparently due to fungal infestation.

4.3.2 In vitro propagation of Diplarrena latifolia

4.3.2.1 Explant source: Seeds

4.3.2.1.1 Disinfestation The results for disinfestation of D. latifolia seeds, including the number of seeds that germinated, have already been described in Chapter 2 (section 2.3.2.2.1).

4.3.2.1.2 Multiplication After 1 months growth on the 10 multiplication media treatments D. latifolia shoots had multiplied on all media tested, with the addition of all cytokinins (at all concentrations) increasing the number of shoots produced compared to the control (Fig. 4.4). The greatest proliferation of new shoots occurred when shoots were grown on MS + 32pM BAP and MS + 321AM 2iP, which produced very similar numbers of new shoots, approximately 4.5 times higher than the control (Fig. 4.4).

BAP was the most successful cytolcinin at all concentrations tested, causing shoots to multiply faster than 2iP and kinetin. 2iP was the least successful at the two lowest concentrations, but was very similar to BAP at the highest concentration (Fig. 4.4).

358 Chapter 4. In vitro propagation of Iridaceae species

10

7.5

Mean no. of new -I- shoots

2.5

MS MS MS MS MS MS MS MS MS MS + + + + + + + + + 201 21AM 2p.M 8p.M 8pM 81.1.M 32pM 3201 320 BAP 2iP Kin BAP 2iP Kin BAP 2iP Kin Treatment

Figure 4.4. The effect of different concentrations of the cytokinins BAP, 2iP and kinetin (Kin) on the mean number of new shoots produced by D. latifolia shoots after 1 month on multiplication media.

Following the first subculture (approximately 3 months after shoots were placed on multiplication media), the mean number of new shoots produced by shoots had increased in all treatments, but, with the exception of BAP and 2iP changing places at the highest concentration of cytokinin, the pattern of the graph had not changed from the results obtained one month after placement on the multiplication media (Fig. 4.5). The ANOVA showed that there were significant differences between the multiplication media treatments (Table 4.8). The highest mean number of new shoots (26.23) occurred on shoots growing on MS + 321.tM 2iP (Fig. 4.5). This treatment was significantly different to all other treatments (Table 4.9). Very high mean numbers of new shoots also grew on the MS + 32[LM BAP (21.40) and MS + 321.1M kinetin (18.17) treatments (Fig. 4.5). These treatments were also significantly different to all other treatments (Table 4.9). The lowest mean number of shoots (3.13) grew on the control treatment (Fig. 4.5). All other treatments produced

359 Chapter 4. In vitro propagation of Iridaceae species significantly higher mean shoot numbers, with the exception of the MS + 21.1M concentrations of all cytokinins tested (Table 4.9).

30

20

Mean no. 15 of new shoots 10

MS MS MS MS MS MS MS MS MS MS + + + + + + + + + 2p.M 21.LM 2p.M 81.IM 81.M 81.M 32 pM 3211M 32iiM BAP 21P Kin BAP 2iP Kin BAP 2iP Kin Treatment

Figure 4.5. The effect of different concentrations of the cytokinins BAP, 2iP and kinetin (Kin) on the mean number of new shoots produced by D. latifolia shoots after 3 months on multiplication media.

Table 4.8. Analysis of Variance for mean number of new shoots produced by D. latifolia shoots on multiplication media.

Source Sum of Squares Df Mean Square F-Ratio P-N a I ue MAIN EFFECTS A: Treatment 17178.137 9 1908.682 85.468 0.000 B: Replicate 43.680 1 43.680 1.956 0.163 RESIDUAL 6453.953 289 22.332 TOTAL (CORRECTED) 23675.770 299

360 Chapter 4. In vitro propagation of Iridaceae species

Table 4.9. Tukey's HSD test groupings of D. latifolia multiplication media treatments (Method: 95.0 percent HSD). Values followed by the same letter are not significantly different (p = 0.05).

Treatment Mean

1. MS (Control) 3.13 a

2. MS + 21.1M BAP 6.53 abc

3. MS + 24.1M 2iP 4.20 ab

4. MS + 21AM kinetin 4.90 ab

5. MS + 811M BAP 11.20d

6. MS + 81.iM 2iP 7.03 bc

7. MS + 81.1.M kinetin 10.10 cd

8. MS + 321.tM BAP 21.40e

9. MS + 321AM 2iP 26.23 f

10. MS + 321.1M kinetin 18.17e

As expected the mean number of new shoots increased with increasing cytokinin concentration for all cytoldnins (Fig. 4.5) (Plate 4.7). For all cytoldnins, the mean shoot number on the 32AM concentration was significantly higher than that on the 81IM and 21.1M concentrations (Table 4.9). The 811M concentrations also produced significantly more shoots than the 21A.M concentrations, with the exception of 2iP (Table 4.9). However, although very high numbers of new shoots were produced by shoots growing on the highest cytokinin concentration (321.11V1), the majority of these shoots had also developed callus. A small percentage of shoots (approximately 5%) also developed callus on the 81.A4 concentration of BAP and 2iP. The majority of shoots growing on the control treatment and the 21AM concentrations of all cytolcinins tested also grew roots (approximately 98% on the control; 85% on all ZAM concentrations). A small proportion on the 81AM concentrations also grew roots (approximately 10% on BAP and 2iP and 15% on kinetin).

361 Chapter 4. In vitro propagation of Iridaceae species

Plate 4.7. D. latifolia shoots after 3 months on multiplication media. Note the low level of multiplication on MS (a) and the very high shoot proliferation on MS + 321aM 2iP (b). Excellent multiplication was also achieved on MS+ 81.1M BAP (c). Tube diameters = 6.5 cm.

4.3.2.1.3 Rooting The majority of shoots from some of the multiplication media treatments produced roots as well as shoots (Table 4.10).

Shoots growing on multiplication media treatments that did not grow roots (Table 4.10) were transferred to MS media. After approximately 1 month on MS media, roots had begun to grow on shoots that were previously growing on all cytokinins at

362 Chapter 4. In vitro propagation of Iridaceae species the 8uM concentration. However, there were still no roots on any shoots previously grown on the 32RM concentrations of all cytokinins. After 2.5 months on MS, shoots initially from the 32IAM concentration of lcinetin began to grow roots, but those previously from the same concentration of 2iP and BAP took longer; only beginning to form roots after 3 months growth on MS.

Table 4.10. The percentage of D. latifolia shoots that also produced roots when growing on each multiplication media treatment. Media treatment Percentage of shoots that grew roots (%) 1. MS (control) 98 2. MS + 2gM BAP 85 3. MS + 2gM 2iP 87 4. MS + 2gM kinetin 84 5. MS + 8gM BAP 10 6. MS + 8gM 2iP 9 7. MS + 8gM kinetin 15 8. MS + 32gM BAP 0 9. MS + 32gM 2iP 0 10. MS + 32gM kinetin 0

After 4 months on MS the percentage of shoots that grew roots was higher for those previously grown on the 81.LM concentration of all cytokinins, than the 321.4.M concentration (Table 4.11).

Table 4.11. The percentage of D. latifolia shoots that produced roots after 4 months growth on basal MS. Media treatment Percentage of shoots that grew roots (%) 1. MS (control) N/A 2. MS + 204 BAP N/A 3. MS + 2gM 2iP N/A 4. MS + 2gM kinetin N/A 5. MS + 8gM BAP 66 6. MS + 8gM 2iP 62 7. MS + 8gM Kinetin 75 8. MS + 3204 BAP 22 9. MS + 32gM 2iP 34 10. MS + 32gM Kinetin 38

363 Chapter 4. In vitro propagation of Iridaceae species

4.3.2.1.4 Transplanting Transplanting was initially quite successful, with 97% of plantlets still alive 1 month after their removal from tissue culture conditions. However, 2 months later, only 68% were still alive. Those that were dead had rotted, apparently due to fungal infestation.

4.3.2.2 Explant source: Whole immature flower buds

4.3.2.2.1 Disinfestation and Initiation The disinfestation procedure was successful with 100% of flower bud explants remaining free of any contaminating organisms approximately 2 months after cultures were initiated. Shoots began to regenerate from 12.5% of stage 1 buds after approximately 2 months in vitro (Table 4.12; Appendix 4.1). At this time, there was no regeneration from any buds at other stages of development (Table 4.12), and quite a high proportion had turned brown and appeared to be dead, or dying (Appendix 4.1).

Table 4.12. The percentage of D. latifolia flower buds that had regenerated shoots (from each developmental stage) after 2 months growth on FBM.

Developmental stage Percentage of flower buds that had regenerated shoots (%) 1 12.5 2 0 3 0

A further 2 months later, the stage 2 buds appeared to be the most regenerative, while there was still no regeneration from any of the youngest, stage 3, buds (Table 4.13; Appendix 4.2). Some of the oldest buds had begun to develop seed capsules, and more explants had died (Appendix 4.2). The stage 1 bud produced 9 shoots, while the stage 2 buds only produced 1 shoot each.

364 Chapter 4. In vitro propagation of Iridaceae species

Table 4.13. The percentage of D. latifolia flower buds that had regenerated shoots (from each developmental stage) after 4 months growth on FBM. Developmental stage Percentage of flower buds that had regenerated shoots ('%) I 12.5 _' 25 3 0

4.3.2.2.2 Multiplication The majority of shoots multiplied on the FBMM, with those originally from the oldest buds (stage 1) producing approximately twice the mean number of shoots that the intermediate (stage 2) buds did (Fig. 4.6). A maximum of 7 new shoots were produced by each shoot from Stage 1, and 2 by Stage 2 shoots. Unfortunately due to such a small number of shoots being originally produced from the flower bud explants, the multiplication data could not be analysed statistically.

Figure 4.6. The mean number of new shoots produced by D. latifolia shoots originally growing from flower bud explants at two different stages of development.

365 Chapter 4. In vitro propagation of Iridaceae species

4.3.3 In vitro propagation of Isophysis tasmanica

4.3.3.1 Explant source: Seeds

4.3.3.1.1 Disinfestation The disinfestation procedure was very successful, with 100% of seeds remaining free of contamination.

4.3.3.1.2 Multiplication

4.3.3.1.2.1 Initial multiplication media experiment (for increasing shoot numbers)

Approximately 5.5 months later, the number of seedlings that had produced new shoots, the number of seedlings that hadn't produced any new shoots, and the mean number of new shoots produced by seedlings on each media treatment were determined (Table 4.14). Seedlings on media containing 21.1M BAP and 21AM 2iP performed better than those on the 21.LM kinetin treatment. Only 13% of seedlings on this media produced new shoots, with an average of 1 new shoot per seedling, compared to 35% and 45%, with an average of 9.75 and 11.4 new shoots, for the 2p.1■4 BAP and 211M 2iP treatments respectively (Table 4.14).

Table 4.14. The percentage of Isophysis tasmanica seedlings that produced new shoots, percentage that didn't produce new shoots, total and mean number of new shoots produced by seedlings grown on each media treatment.

Media treatment Percentage Percentage Total Mean Percentage of seedlings of seedlings number number of dead with new without new of new of new seedlings shoots (%) shoots (%) shoots shoots (%) 1. MS + 21.tM kinetin 13 69.6 3 1 14.8 2. MS + 21.1M BAP 34.8 65.2 78 9.75 20.7 3. MS + 21.1.M 2iP 45 55 103 11.4 0

At the time of subculturing to MS + 41M BAP, the mean number of new shoots produced was highest for the MS + 2pLM 2iP treatment (Table 4.15). The multiplication of shoots in the treatment containing kinetin was still very low (Table 4.15).

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Table 4.15. The mean number of shoots produced by I. tasmanica seedlings on the different initial multiplication media, at the time of subculture to MS + 21.1.M BAP media.

Previous media Initial number of Total number of Mean number of treatment seedlings (before shoots (following shoots produced subculture) subculture) per seedling 1. MS + 21AM kinetin 13 24 1.85 2. MS + 21AM BAP 21 126 6.00 3. MS + 21.J.M 2iP 20 154 7.70

At the time of transfer to the MS + AC treatment, 4.5 months after shoots were placed on MS + 21AM BAP, the mean number of new shoots produced followed the same pattern as that before transfer to MS + 21.tM BAP (Table 4.16). However, the transfer to the new media increased the mean number of new shoots produced by shoots in all treatments.

Table 4.16. The mean number of new shoots produced by I. tasmanica shoots on the different initial multiplication media, at the time of subculture to MS + AC.

Previous media Initial Number of Vaal Mean number treatment number of dead or number of of new shoots shoots contaminated shoots produced per (before shoots (following surviving shoot subculture) subculture) 1. MS + 21iM kinetin 24 7 dead 49 2.88 then MS + 211,M BAP 2. MS + 21AM BAP 126 25 dead 727 7.57 then MS + 21.t.M BAP 5 contam. 3. MS + 2iiM 2iP 154 1 dead 1416 10.04 then MS + 21.1.M BAP 12 contam.

4.3.3.1.2.2 Multiplication media experiment

The ANOVA showed that there were significant differences between multiplication media treatments (Table 4.17). Shoot multiplication occurred in all treatments, with the highest number of new shoots produced by shoots grown on MS + 32IAM BAP (Fig. 4.7). However, this treatment was not significantly different to two other treatment means (for MS + 8RM BAP and MS + 32gM kinetin) (Table 4.18). These

367

Chapter 4. In vitro propagation of Iridaceae species three treatments produced significantly more shoots than all other treatments (Table 4.18).

Table 4.17. Analysis of Variance for mean number of new shoots produced by I. tasmanica shoots on multiplication media.

Source Sum of Squares Df Mean Square F-Ratio P-N alue MAIN EFFECTS A: Treatment 1266.658 15 84.444 23.078 0.000 B: Replicate 1.786 1 1.786 0.488 0.485

RESIDUAL 1694.148 463 3.659 TOTAL (CORRECTED) 2962.592 479

1 0

j. I 7.5 —

Mean no. of new shoots 727 2.5 —

mama • MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS + + + + + + + + + + + + + + + 0.5pM 2pM 8pM 32 pM 128 p.M 0.50 2pM 8pM 32 pM128gM 0.5pM 2pM 8pM 320 1280 Kin Kin Kin Kin Kin 21P 2iP 2iP 2iP 2iP BAP BAP BAP BAP BAP

Treatment

Figure 4.7. The effect of different concentrations of the cytokinins kinetin (Kin), 2iP and BAP on the mean number of new shoots produced by I. tasmanica shoots.

The control treatment, which contained no PGRs, actually produced a higher mean number of new shoots than shoots grown on three of the cytokinin treatments (Fig. 4.7) However, differences were not significant (Table 4.18). Roots also grew in all replicates in the control treatm-ent. There was a general trend of increasing shoot numbers with increasing cytokinin concentration in the media for all cytokinins (Plate 4.8). However, this trend did not continue above the 32AM concentration, with the highest concentration tested (128AM) actually causing a decrease in the number

368 Chapter 4. In vitro propagation of Iridaceae species of new shoots produced, and the development of callus (Plate 4.9). That decrease in shoot number was to a lower number of shoots than that produced by shoots growing on the lowest concentration (0.51.1M) in the case of kinetin and BAP (n.$) (Table 4.18).

Table 4.18. Tukey's HSD test groupings of I. tasmanica multiplication media treatments (Method: 95.0 percent HSD). Values followed by the same letter are not significantly different (p = 0.05). Treatment Mean 1. MS (Control) 3.90 ab 2. MS + 0.5uM kinetin 4.50 abc 3. MS + 21.tM kinetin 4.17 abc 4. MS + 8uM kinetin 5.60 c 5. MS + 32p,M kinetin 7.80 d 6. MS + 128uM kinetin 3.20 a 7. MS + 0.511M 2iP 3.30a 8. MS + 2uM 2iP 3.20 a 9. MS + 81.1.M 2iP 5.10 bc 10. MS + 32uM 2iP 5.77 c 11. MS + 124LM 2iP 5.07 bc 12. MS + 0.51.1M BAP 4.77 abc 13. MS + 21.1.1%.4 BAP 5.33 bc 14. MS + 8mM BAP 7.97 d 15. MS + 32uM BAP 8.57d 16. MS + 128uM BAP 4.23 abc

The most successful cytokinin, in terms of the mean number of shoots produced, was BAP. The number of new shoots was highest for shoots growing on BAP at all concentrations. At 0.5uM concentrations, BAP produced more shoots than 2iP and kinetin (n.s., Table 4.18). At 21.1.M concentrations, shoots growing on BAP produced significantly more shoots than those growing on 2iP, but not kinetin (Table 4.18). At 81AM concentrations, shoots growing on BAP produced significantly more shoots than those growing on both 2iP and kinetin (Table 4.18). At 3211M concentrations, shoots growing on BAP produced significantly more shoots than those growing on 2iP, but not kinetin (Table 4.18), while, at the highest concentration (1281.1M), BAP produced higher numbers of shoots than kinetin (n.s., Table 4.18), but the mean shoot number was lower than that produced by shoots growing on 12811M 2iP (n.s, Table 4.18).

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Plate 4.8. L tasmanica shoots growing on multiplication media containing increasing concentrations of the cytokinin BAP: MS — no BAP (a), MS + 8uM BAP (b), and MS + 3211M BAP (c). Tube diameters = 6.5 cm.

Plate 4.9. I tasmanica shoots growing on MS + 12811M BAP. Note the development of callus (a), and shoots regenerating from the callus (b). Tube diameter = 6.5 cm.

370 Chapter 4. In vitro propagation of Iridaceae species

4.3.3.1.3 Rooting Approximately 87% of shoots grew roots on the MS + 1g1: 1 AC media, therefore a rooting media trial was not required.

4.3.3.1.4 Transplanting One month after transplanting to ex vitro conditions, 76% of the Isophysis tasmanica plantlets had survived. Those that died appeared to have rotted at the bases.

Three months after transplanting, the percentage of surviving plants had decreased to only 56%, with the majority of plants that died being apparently due to rotting caused by fungal infestation.

Twelve months after plants were repotted, only 26% of those originally transplanted were still alive.

4.3.3.2 Explant source: Whole immature flower buds Two days after the I. tasmanica flower buds were placed on FBM, 81.8% of the buds had opened and all were free of contamination.

Twenty one days after cultures were initiated the percentage of opened flower buds had not changed, and all of these explants were either already brown or beginning to turn brown. There was no sign of any regeneration at this stage.

Approximately 1 month after cultures were initiated, the rest of the buds had opened, and all were dead. There was no regeneration from any of the explants.

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4.4 Discussion

4.4.1 In vitro propagation of Diplarrena moraea

4.4.1.1 Explant source: Seeds A complete protocol for the propagation of D. moraea in vitro using seeds as the initial explant source was devised. Although seeds are not an ideal source of explant for micropropagation where clones are the expected outcome, they were the only suitable explant at the time the experiment began. Therefore, in vitro germinated seedlings were used, in the hope that the results from this experiment could be employed for micropropagation of shoots from other explant types in the future.

As expected, D. moraea seeds (which are held in a until dehiscence) were very easy to sterilise (this had already been determined in Chapter 2). Immersion in a 2% Na0C1 solution for 20 min was enough to remove all microorganisms from the surface of all seeds. Disinfestation of above-ground structures, especially those protected by outer coverings, is generally much more successful than below-ground or exposed regions (George, 1996; Bunn and Dixon, 1997; Ziv and Lilien-Kipnis, 2000).

The seedlings, and shoots obtained from them, grew well on MS, which has also been used successfully for growing many other Iridaceae genera in vitro, including Iris (De Munk and Schipper, 1993; Shimizu etal., 1997; Shibli and Ajlouni, 2000), Gladiolus (Hussey, 1975b, 1976, 1980; Ziv, 1979; Jager etal., 1988; Kumar etal., 1999), Freesia (Hussey, 1975b, 1976, 1980; Pierik and Steegmans, 1976; Doi et al., 1992) Crocus (Ding et al., 1979, 1981; Chichiricco and Grilli Caiola, 1987; Homes etal., 1987; Ilahi etal., 1987; Isa and Ogasawara, 1988; Fakhrai and Evans, 1989; George etal., 1992; Milyaeva etal., 1995), Sparaxis and Schizostylis (Hussey, 1975b).

Shoot multiplication was achieved on all multiplication media tested, which suggests that BAP, kinetin and 2iP are all suitable cytokinins for shoot production of this species in vitro. The mean number of new shoots increased with increasing

372 Chapter 4. In vitro propagation of Iridaceae species cytoldnin for BAP and 2iP (only one concentration of kinetin was used), with the greatest shoot proliferation occurring on seedlings growing on MS + 1281AM 2iP. However, as only two different concentrations were used (at the low and high end of the spectrum) it was not possible to ascertain the optimal concentration or the best cytoldnin to use for shoot multiplication of this species. Further experimentation using a wider range of cytokinins is required. The fact that callus was produced by the majority of seedlings growing on the highest cytolcinin concentration (for both BAP and 2iP), also suggests that these treatments are not ideal for this species. Callus should generally be avoided in a micropropagation system where clones are required, as it is often associated with somaclonal variation (Dodds and Roberts, 1985; Stewart, 1999b). However, for some Iridaceae genera, such as Freesia, callus has been found to be genetically stable (Van Aartijk and Van der Linde, 1986) and it is therefore appropriate to use a method in which plantlets are regenerated from it. It may be possible that D. moraea also has genetically stable callus. However, until this is determined any shoots with callus should be avoided.

A relatively small proportion of shoots growing on all multiplication media treatments were dead by the time shoots were subcultured. However, as the proportions were similar across all treatments it is unlikely that the death of shoots was due to any treatment effects such as cytokinin toxicity. Instead it was most likely due to natural attrition, or possibly due to the shoots remaining on the media for too long and having used up the nutrients within the media.

All of the shoots growing on the control treatment and the 21AM concentrations of all cytokinins grew roots as well as shoots, which meant that a separate rooting media was not required. As no root growth occurred on shoots growing on the highest cytokinin concentration (128pM) this suggests that the lower concentrations were unable to inhibit the growth of roots. Being able to transplant rooted plantlets directly from the multiplication media means that the time taken to produce a marketable product can be reduced, which is highly desirable in a commercial situation. However, as these low cytokinin concentrations may not be optimal for shoot production, it is possible that a higher concentration (that may be optimal for shoot multiplication) would inhibit the growth of roots. Therefore, a separate rooting

373 Chapter 4. In vitro propagation of Iridaceae species

media stage may in fact be necessary for D. moraea, and further investigation is required.

The transplanting procedure was initially quite successful, with 100% of plantlets still alive 1 month after their removal from tissue culture conditions. However, 2 months later this percentage had decreased to 63%, with the majority of plants that died succumbing to fungal infestation and rotting. This result shows the importance of employing the correct watering regime. The fungal infestation was almost certainly due to over-watering as the potting mix was waterlogged. Therefore, in future the transplants must be carefully monitored to ensure that they receive the correct amount of moisture to prevent desiccation, but not so much that they become waterlogged.

The results from this experiment show that micropropagation of D. moraea is possible. Although the investigation was only preliminary it has provided clear direction for future research using other more suitable explant sources.

4.4.2 In vitro propagation of Diplarrena latifolia

4.4.2.1 Explant source: Seeds The major expectation and outcome of a micropropagation system is that clones of the original parent plant are produced. Therefore, as mentioned in the previous section, seeds are obviously not an initial explant of choice due to the recombination of DNA that occurs when they are formed. However, as was the case for D. moraea, they were the only available explant at the time the experiment commenced, and had already been germinated for seed germination studies (see Chapter 2). Therefore, in vitro germinated seedlings were used for further micropropagation studies. It was hoped that the results obtained could be employed for micropropagation of shoots from other explant types in the future.

Similar to D. moraea, D. latifolia seedlings and shoots also grew well on MS, and shoot multiplication was enhanced by the addition of all cytokinins to the media. After 1 months growth on multiplication media the greatest shoot proliferation

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occurred when shoots were grown on MS + 321iM BAP, and BAP was the most successful cytokinin at all concentrations tested. BAP also produced higher numbers of new shoots than kinetin and 2iP at the 241M concentration for D. moraea in the previous experiment, which suggests that these closely related species respond similarly in vitro, as was expected.

The results obtained following the first subculture (approximately 3 months after shoots were placed on multiplication media) followed a very similar pattern to those after only 1 month, which shows that the initial results were quite accurate in terms of the best cytokinin and concentration to use. Therefore, in future experiments with this species, accurate results could be obtained quite quickly, which would speed up the experimental process.

As expected, the mean number of new shoots increased with increasing cytokinin concentration for all cytokinins tested. However, as was the case for D. moraea, at the highest concentration, the shoots also developed callus. A very small percentage of D. latifolia shoots growing on the 81.1M concentration of BAP and 2iP also had some callus present. Therefore, for future multiplication work with D. latifolia the best media to use would probably be either MS + 81.IM BAP, with any shoots developing callus being discarded, or MS + 81.1M kinetin, which also produced quite a high number of new shoots without any callus present. It would be wise to avoid the 3211M concentrations unless the callus produced by this species is proven to be genetically stable.

The results obtained from this multiplication media experiment could probably be extrapolated for use with D. moraea as the two species appear to respond similarly in vitro. The mean number of new shoots obtained from both species after 3 months growth on all cytokinins (at a 211M concentration) was very similar.

The majority of D. latifolia shoots growing on MS and MS supplemented with 2IAM concentrations of all cytokinins grew roots as well as shoots; which was also the case for D. moraea. Again showing the similarity in in vitro response of these closely related species. There was also some root development on a small proportion of shoots growing on the 811M concentration of all cytokinins. Therefore, there was no

375 Chapter 4. In vitro propagation of Iridaceae species need for a separate rooting media experiment, and shoots with roots were transplanted to ex vitro conditions. However, as only a low percentage of shoots growing on the best multiplication media (MS + 81.1M BAP, see above) grew roots it was thought prudent to remove these shoots from multiplication media and place them on a basal media. By removing the cytokinin from the media it was hoped that explants would be able to develop roots. This did occur, with roots developing quite quickly on shoots previously grown on all cytokinins at an 81.1M concentration. However, at the higher 321.1M concentration shoots took considerably longer. The percentage of shoots that developed roots was also higher for shoots previously grown on the lower concentration (8pM) of all cytokinins. This suggests that, even though shoots were removed from the media supplemented with cytokinin, it still took time for the residual effects to be removed. Taking longer for the higher concentration than the lower, as would be expected. Therefore, it would still be worthwhile to conduct a separate rooting media experiment, as a medium containing auxin may enhance and speed up the development of roots compared to the method of removing them from multiplication media and placing them on basal MS, used in the present experiment.

Shoots that developed roots on the multiplication media initially survived well when transplanted to ex vitro conditions. Only 3% had died one month later, which was an excellent result. However, survival had decreased dramatically three months after transplanting. This also occurred with D. moraea transplants, and again the majority that died had succumbed to fungal infestation and rotting. This result reiterates the importance of the correct watering regime for such fragile plants. As was suggested for D. moraea, in future transplants must be carefully monitored to ensure that they receive the correct amount of moisture to prevent desiccation, but not so much that they become waterlogged.

This experiment showed that micropropagation of D. latifolia was possible, and that it responds in a similar way to the closely related D. moraea. An entire protocol, beginning with seeds as the explant source was determined, and rooted plantlets were successfully transplanted to ex vitro conditions. It is believed that this protocol can be used with another more suitable explant type to ensure that clones are produced.

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4.4.2.2 Explant source: Whole immature flower buds The use of immature inflorescence parts as an explant source has been successful for a wide range of monocots including the Iridaceae family. Inflorescence stem sections have been used for in vitro propagation of Iris (Meyer etal., 1975; Hussey, 1976b; Meyer, 1984; Bach, 1988), Gladiolus (Ziv etal., 1970; Simonsen and Hildebrandt, 1971; Hussey, 1975b, 1976; Kumar etal., 1999) and Freesia (Hussey, 1975a,b; Wang et al., 1994); peduncle tissue, still enclosed by the spathe, has been successful for Gladiolus (Ziv and Lilien-Kipnis, 2000), while flower buds have been used to initiate cultures of Freesia (Pierik and Steegmans, 1975, 1976). Many other monocots have been propagated in vitro using flower buds as initial explants, including the Liliaceae species mentioned in the previous chapter, the Australian native Anigozanthos (Oliver etal., 1996) and preliminary work appears promising for flower bud explants of Doryanthes excelsa (Smith, 2000).

Floral explant types have many advantages over the use of subterranean organs. They are easier to sterilise (Slabbert et al., 1995; George, 1996; Amomarco and Ibanez, 1998; Ziv and Lilien-Kipnis, 2000) as they are often held within protective outer coverings and are generally free of microbial contaminants (George, 1996). Their removal from the parent plant is also less invasive, meaning that the storage and perennating organ of the mother plant remains intact for further growth and selection (Ziv and Lilien-Kipnis, 2000). Floral explants have also been found to be more regenerative than other explant types associated with the storage organ. For example, in experiments with Gladiolus grandiflorus 10 buds developed on a 3 to 4 mm inflorescence explant, compared to only 3 on apical bud explants (Ziv and Lilien-Kipnis, 2000). Therefore, flower buds were selected as a more suitable explant than the seeds used in the previous section, to try for D. latifolia.

As expected, D. latifolia flower buds were very easy to sterilise, with 100% of explants remaining free of any contaminating organisms. However, in terms of regeneration from the explants, results were promising, but not overly successful. After 2 months in vitro shoots had regenerated from only 1 bud, which represented 12.5% of stage 1 buds. It was unusual that the most mature buds were initially the most regenerative, as younger tissues generally regenerate better than older, more

377 Chapter 4. In vitro propagation of Iridaceae species

mature tissues (Hartmann and Kester, 1983). Quite a high proportion of buds from all developmental stages had turned brown and appeared to be dead or dying, which perhaps suggests that they were either: (i) not at the correct stage of development, (ii) that the media was not optimal for the growth of this explant type, or (iii) the fact that they turned brown may indicate the presence of phenolic compounds in this species. This is not unlikely as many monocots have been found to contain high levels of these compounds (Taji and Williams, 1996; Zaid, 1987; Panaia, 1998), including Doryanthes excelsa, from the Doryanthaceae which has distant links with the Iridaceae (M. Fay, pers.comm. cited in Smith, 2000). Dianella, from the closely related Liliaceae has also been reported to contain phenolics (Jassim and Naji, 2003).

After 4 months on FBM the most regenerative explants were now from stage 2. The percentage of buds that had regenerated shoots at this stage of development was quite low (25%), but was twice that of the stage 1 buds (12.5%). Interestingly, even though stage 1 explants were the least regenerative in terms of the percentage of explants that developed shoots, the number of shoots produced by the stage 1 bud was 9 times higher than the stage 2 buds. Therefore, although stage 1 only had a low percentage of explants that developed shoots, the number of shoots produced by explants was actually quite high, suggesting that individual explants at this stage of development had a high capacity for regeneration. However, as explant numbers were so low in this experiment it is difficult to draw accurate conclusions from it.

There was no regeneration at all from any of the youngest (stage 3) buds. The youngest buds from the punicea experiments (see sections 3.3.1.2.1 and 3.3.1.3.1) were also found to be less regenerative than the intermediate and older stages, although some explants did regenerate shoots. For B. punicea the younger buds did not begin to regenerate shoots until 2.5 months later than the stage 1 and 2 buds (see section 3.3.1.3.1). Therefore, had the D. latifolia experiment gone on longer than it did, some regeneration from these buds may have occurred.

The fact that some of the oldest (stage 1) buds had begun to develop seed capsules and a high proportion of explants died suggests that explants were not at the correct stage of development for regeneration to occur. Immature fruit also developed on some Dianella tasmanica flower buds (see section 3.3.2.5.1.1), which led to the

378 Chapter 4. In vitro propagation of Iridaceae species suggestion that if buds were developed enough to produce fruit, then they were perhaps getting too old to regenerate shoots. It should also be noted that, as was the case for D. tasmanica, when capsules (or fruit in the case of D. tasmanica) were produced this occurred from previously unopened buds, and therefore fruit had apparently formed by apomixis. This phenomenon had also been noticed in mature field-grown plants of D. tasmanica (Curtis, 1952; Sward, personal observation). It is not known whether it has been noticed in D. latifolia.

Shoots produced by D. latifolia flower bud explants multiplied well on FBMM, which suggests that it is an appropriate medium to use. The number of shoots produced appeared to be influenced by the stage of development of the original explants. Shoots originally growing from stage 1 buds produced approximately twice the mean number of shoots than the intermediate buds (stage 2) did. Initially, they had regenerated 9 times the amount of shoots as the stage 2 buds did.

This preliminary experiment showed that shoot regeneration from flower bud explants of D. latifolia was possible. However, more work is required to ascertain the correct stage of development to subsequently produce the greatest number of shoots. The multiplication media previously used for Blandfordia grandiflora (Bunn and Dixon, 1997) and the Liliaceae species studied in this thesis (Chapter 3) was also suitable for shoot proliferation of D. latifolia. However, in future experiments it may be worthwhile to try multiplying the shoots regenerated from flower buds on MS + 81.1M BAP or MS + 81.IM kinetin. When D. latifolia shoots (originally from seedling explants) were grown on these treatments, higher mean numbers of new shoots were produced than by those grown on FBMM in the current experiment (11.2 and 10.1 for MS + 81.IM BAP and MS + 81.1M kinetin, respectively; cf. 3.2 and 1.5 for explants originally from stage 1 and stage 2 buds, respectively, on FBMM).

379 Chapter 4. In vitro propagation of Iridaceae species

4.4.3 In vitro propagation of Isophysis tasmanica

4.4.3.1 Explant source: Seeds Isophysis tasmanica was initially propagated in vitro using seeds as an explant source. Even though seeds are not really a suitable explant for a micropropagation system where clones are the expected outcome, they were the only available explant at the time the experiment began. I. tasmanica has very short rhizomes and it was impossible to find any pieces with buds. There were also no young shoots developing, so these could not be used either.

Seeds were very easy to sterilise with a simple disinfestation procedure that had already been determined (see section 2.2.3.3.1). There was no contamination of any explants at all. This result was expected as there was no contamination of seeds in a previous in vitro germination experiment (see section 2.3.2.3.1).

To obtain enough shoots for a large scale multiplication media experiment, seedlings were initially grown on three different media treatments containing 211M concentrations of BAP, 2iP and kinetin. Shoot multiplication was much higher on BAP and 2iP than kinetin. BAP was also the most successful cytolcinin, in terms of the number of shoots produced, for both D. moraea and D. latifolia. However, 2iP produced similar shoot numbers as kinetin for both of these species.

Shoots were subcultured after 5.5 months and all were placed on MS + 211M BAP to further increase numbers. The mean number of new shoots produced per shoot increased following subculture, even for shoots previously grown on the same media, which suggests that the subculturing process played a part in increasing the number of new shoots produced. This phenomenon was also noticed in Dianella tasmanica cultures (section 3.3.2.3.1). The initial medium that shoots were grown on also appeared to influence the number of new shoots produced, as those initially grown on kinetin still had the lowest mean number of shoots following subculture and growth on MS + 2gM BAP. Also, those initially grown on MS + 21.1M 2iP still had the highest mean number of new shoots. This is perhaps due to a carry-over of the original cytokinin to the new media.

380 Chapter 4. In vitro propagation of Iridaceae species

Following the initial build-up of shoot numbers they were grown on a MS + 1g1: 1 AC media for 1 month to remove any cytokinins from the media and therefore prevent such carry-over effects for the large scale multiplication media experiment. Sixteen different multiplication media were tested. The most successful in terms of mean number of new shoots produced was MS + 32uM BAP. This treatment was also very successful for D. latifolia, producing the second highest number of new shoots. The fact that shoots growing on the control treatment produced a higher mean number of shoots than those grown on MS + 0.511M 2iP and MS + ZAM 2iP (n.s.) can probably be explained by the fact that these were only very low concentrations of cytokinin, and the shoot numbers were only slightly lower. While those growing on MS + 12811M kinetin probably produced less shoots than the control (n.s.) because this concentration was very high and adversely affected the shoots. Shoots growing on the same concentration of the other two cytokinins also produced less shoots than those growing on lower concentrations, which supports this theory.

BAP was the most successful cytokinin for I. tasmanica, with the number of new shoots produced being highest in shoots growing on this cytokinin at all concentrations tested. It was also the most successful for D. latifolia at the two lowest concentrations (2pM and 8uM) and D. moraea at the 21.jM concentration (only two concentrations were tested for this species).

Eighty seven percent of I tasmanica shoots grew roots on MS + 10: 1 AC, which meant that a rooting media trial was not required. Similar percentages of the Liliaceae species Milligania densiflora (78%) and Dianella tasmanica (71%) also developed roots on media containing AC. Some researchers believe that AC causes plants to grow roots because it darkens the media, thus making conditions similar to growing in soil (Proskauer and Berman, 1970).

Transplanting I. tasmanica rooted plantlets to ex vitro conditions was initially quite successful. Seventy six percent of plantlets were still alive one month after transplanting, which was quite good, but not as successful as the other Iridaceae species (100% of D. moraea and 97% of D. latifolia plantlets were still alive one month after transplanting). I. tasmanica transplants were also adversely affected by

381 Chapter 4. In vitro propagation of Iridaceae species over-watering and fungal infestation. However, this species was more sensitive than the two Diplarrena species, with only 56% of transplants surviving to the 3 month post-transplant stage. Therefore, in future experiments, I. tasmanica transplants must be monitored very carefully to ensure that they don't become waterlogged, while still receiving enough water to prevent desiccation. The sensitivity and difficulty of growing this species is further reiterated by the fact that 12 months after the surviving transplants had been repotted for the first time, only 26% were still alive. It requires excellent drainage or rotting will quickly take place if plants become too wet.

This experiment showed that I tasmanica could be gown in vitro using seeds as initial explants. Shoots multiplied best on MS + 32gM BAP; and these shoots were induced to form roots on MS + 1g1: 1 AC. Transplanting was initially quite successful, but this species appears particularly susceptible to rotting if the soil becomes too wet, so they must be very carefully monitored in future micropropagation experiments. As seeds are not an ideal choice of explant for micropropagation, it is hoped that the protocols devised from this experiment can be used with another more suitable explant type to grow this species in vitro.

4.4.3.2 Explant source: Whole immature flower buds Due to the limited success achieved with D. latifolia flower buds and the unavailability of any other potential explant source, flower buds were tried as an alternative to seeds for I. tasmanica.

Like the D. latifolia flower buds, I. tasmanica buds were also easy to disinfest, with 100% remaining contaminant-free for the course of the experiment. However, none of the buds showed any signs of regeneration and they were therefore unsuccessful. As the majority of the buds had opened within two days of placement in vitro this suggests that the buds were actually quite mature and probably close to opening on the plant before they were removed. The petals already had their colour, which was another sign that they were quite mature. Within 21 days all of the explants were either brown or beginning to turn brown, which also suggests that they were not at the correct stage for regeneration and/or that the media was adversely affecting the

382 Chapter 4. In vitro propagation of Iridaceae species buds. Alternatively, the tissue browning may have been due to phenolic compounds (as discussed previously for D. latifolia flower buds).

The fact that I. tasmanica flower buds did not respond well to in vitro conditions was most likely because they were already too mature. Therefore, this explant type should not be dismissed, but should be investigated further using a range of (less mature) buds. The developmental stage can be crucial to determining the success or otherwise of tissue cultures (Guerra and Handro, 1998).

4.5 Summary and Future Research

4.5.1 Diplarrena moraea • Seeds were used as an initial explant source for Diplarrena moraea.

• 100% of D. moraea seeds were contaminant free following a 20 min sterilisation period in 2% Na0C1.

• The mean number of new shoots produced by D. moraea subcultured seedlings increased with increasing cytokinin concentration in the media.

• D. moraea seedlings produced very high numbers of new shoots on the media supplemented with 128uM BAP and 1281A4 2iP. These treatments produced significantly more shoots than the control.

• The majority of D. moraea seedlings growing on the highest cytokinin concentration (128mM) on both BAP and 2iP also developed callus.

• D. moraea seedlings growing on the control (MS) and 2uM concentrations of BAP, kinetin and 2iP also grew roots.

• A proportion of shoots from all treatments were dead at the time shoots were subcultured.

• The proportion of dead shoots was similar for all treatments and not significantly different.

383 Chapter 4. In vitro propagation of Iridaceae species

• As all of the shoots growing on treatments containing low concentrations of, or no (in the case of the control) cytokinin, also grew roots a separate rooting medium was not required.

• Transplanting to ex vitro conditions was initially very successful - 100% were still alive 1 month later.

• After 3 months the proportion of surviving plantlets had decreased to 63%.

• The majority of plantlets that died had rotted due to fungal attack.

As seeds are not an ideal explant source for micropropagation, where clones of the original plant are generally required, other explant sources should be investigated for D. moraea. Due to the limited success achieved using immature flower bud explants for the closely related D. latifolia, this would be an explant to pursue for D. moraea. Other floral explants, such as pedicel/peduncle junctions and sections of the scape, which were very successful for some of the Liliaceae species in this study would also be worth trying.

The multiplication media experiment was very limited in the present study, with only two concentrations at high and low ends of the range used for BAP and 2iP, and only one low concentration of kinetin. Therefore, in future experiments a wider range of concentrations should be used to determine the optimal concentration of the best cytokinin for shoot multiplication of this species. However, as a wide range of concentrations of the three cytokinins (BAP, kinetin and 2iP) were tested on the closely related D. latifolia it may be expected that D. moraea would respond similarly. In terms of rooting, although roots formed on shoots growing on many of the multiplication media tested, it may be worthwhile to test the development of roots on a range of media specifically formulated for the development of roots. A specifically designed rooting media may increase the number of roots produced which could be beneficial at transplanting.

384 Chapter 4. In vitro propagation of Iridaceae species

4.5.2 DiplatTena latifolia

• Seeds were the first explant source chosen for D. latifolia.

• Shoots obtained from D. latifolia seedlings (germinated in vitro) were used for a multiplication media experiment.

• After 1 month the addition of all cytokinins tested (BAP, 2iP and kinetin) had increased the number of new shoots compared to the control.

• The greatest proliferation of new shoots occurred when shoots were grown on MS + 321.IM BAP and MS + 321AM 2iP.

• BAP was the best cytokinin at all concentrations tested (after 1 month), causing shoots to increase in number faster than 2iP and kinetin.

• Following the first subculture the mean number of new shoots had increased in all treatments.

• MS + 3212M 2iP had the highest number of new shoots following the first subculture. The mean number of new shoots produced by shoots on this treatment was significantly different to those growing on all other treatments.

• Very high mean shoot numbers also occurred on shoots growing on MS + 321AM BAP and MS + 32tiM kinetin. These results were also significantly different to those of all other treatments.

• The mean number of new shoots increased with increasing cytokinin concentration in the media, for all cytokinins (BAP, 2iP and lcinetin).

• For all cytokinins the mean shoot number on the 321.1M concentration was significantly higher than that on the 81.1.M and ZAM concentrations.

• The 81AM concentrations of all cytokinins (with the exception of 2iP) also produced significantly more shoots than the 2pM concentrations.

• The majority of shoots growing on the highest concentration of all cytokinins (321iM) also developed callus.

385 Chapter 4. In vitro propagation of Iridaceae species

• A small percentage (— 5%) also developed callus on the 81.IM concentration of BAP and 2iP.

• The majority of shoots growing on the control treatment and the 21.LM concentration of all cytokinins tested grew roots as well as shoots on the multiplication media.

• A small proportion of shoots also grew roots on the 8tiM concentration of all cytokinins.

• Shoots that did not grow roots on the multiplication media were transferred to basal MS media.

• After approximately 1 month on MS, shoots previously grown on the 81.1M concentration of all cytokinins began to grow roots.

• Shoots previously grown on the 321aM concentration of all cytolcinins took longer - 2.5 months for kinetin and 3 months for 2iP and BAP.

• After 4 months on basal MS media the percentage of shoots that grew roots was higher for those previously grown on the 81.LM concentration (of all cytokinins) than the 321.xM concentration.

• More shoots, previously grown on kinetin (at 8W and 32A4 concentrations), developed roots on MS than those previously on 2iP and BAP (at the same concentrations).

• Transplanting to ex vitro conditions was initially very successful, with 97% of plantlets still alive 1 month after removal from tissue culture conditions.

• Three months after transplanting, survival had decreased to 68%.

• The majority of dead plants had rotted due to fungal attack.

• Immature flower buds were also used as an initial explant source for D. latifolia.

• D. latifolia flower bud explants were easy to disinfest with 100% of explants remaining sterile after 2 months in vitro.

386 Chapter 4. In vitro propagation of Iridaceae species

• Shoots began to regenerate from the most mature (stage 1) buds first, after approximately 2 months in vitro.

• After 4 months in vitro the most regenerative explants were the intermediate (stage 2) buds - 25% of these had regenerated shoots.

• There was no shoot regeneration from the youngest (stage 3) buds at all.

• Quite a high proportion of explants from each developmental stage turned brown and died in vitro.

• The stage 1 bud produced the highest number of shoots (9 cf. 1 for the stage 2 buds).

• The majority of the shoots (produced by flower buds) multiplied on FBMM.

• Shoots originally from the oldest (stage 1) buds produced twice the mean number of shoots that the intermediate (stage 2) shoots did.

As mentioned in the previous section, seeds are not an ideal explant source for micropropagation. Therefore, the preliminary research into the use of flower bud explants would be more appropriate to pursue. Classification of flower buds into different stages based on size would perhaps enable the best growth stage for future shoot regeneration to be determined more accurately. A much wider range of developmental stages should also be included in future experiments as it is possible that the optimal stage may have been missed in the current study. As mentioned for D. moraea other floral explant types such as pedicel/peduncle junctions and scape sections should also be assessed for their ability to regenerate shoots, as it is possible that they may yield greater numbers of shoots than the flower bud explants. It would also be worthwhile trying a different initiation media, such as MS + 5pM NAA and lOpM lcinetin, which was the most successful media for shoot induction on peduncle tissue of Gladiolus grandiflorus (Iridaceae) (Ziv and Lilien-Kipnis, 2000).

In terms of shoot multiplication, higher numbers of new shoots were produced by shoots on the majority of multiplication media treatments, than FBMM. Therefore, it may be worthwhile to multiply shoots (regenerated on flower bud explants) on one of the other multiplication media (eg. MS + 8pM BAP or MS + 8pM kinetin).

387 Chapter 4. In vitro propagation of Iridaceae species

For rooting, although a separate rooting media was not required (as roots developed on shoots in some of the multiplication media treatments) it may be worthwhile to still do a separate rooting media trial, as this may hasten the development of roots and/or increase the number of roots produced, which may be beneficial after transplanting.

The high proportion of flower bud explants that turned brown and died may have been due to oxidation of phenolic compounds within the tissues. Therefore, the use of a pre-culture wash with an antioxidant treatment (eg. potassium citrate and citrate) would be worth trying.

Somatic embryogenesis should also be investigated as a means of mass propagation of both Diplarrena species. It has been achieved for many ornamental flower bulb and corm genera in the Iridaceae family, eg. Iris (Reuther, 1977; Shimizu etal., 1996, 1997; Radojevic etal., 1987; Radojevic and Landre, 1990; Laublin et al., 1991; Radojevic and Subotic, 1992; Jehan et al., 1994; Wang et al., 1999; Shibli and Ajlouni, 2000), Freesia (Wang etal., 1994) and Crocus (George et al., 1992; Karamian and Ebrahimzadeh, 2001) and may also be possible for native Australian Iridaceae species.

4.5.3 Isophysis tasmanica

• Seeds were the first explant source tried for I tasmanica.

• The disinfestation procedure was very successful, with 100% of seeds remaining free of contamination.

• Seedlings were initially multiplied on three different media: MS + 21.tM BAP, MS + 21.1M kinetin and MS + 41M 2iP.

• Shoot multiplication was much higher on BAP and 2iP than kinetin.

• A large scale multiplication media experiment that included 16 different treatments was performed.

388 Chapter 4. In vitro propagation of Iridaceae species

• Shoots growing on MS + 3211M BAP produced the highest number of new • shoots. This treatment produced significantly more shoots than the majority of other treatments (with the exception of MS + 81.1M BAP and MS + 321.tM kinetin).

• Shoots growing on MS + 81.1M BAP and MS + 321AM kinetin also produced high numbers of new shoots. The results from these two treatments were also significantly different to the other treatments (with the exception of each other and MS + 321AM BAP).

• Shoots growing on the control treatment produced higher numbers of new shoots than three of the treatments containing cytokinin (MS + 128pM kinetin, MS + 0.511M 2iP and MS + 21.LM 2iP) (n.s.).

• Between 0.51AM and 32gM concentrations of all cytokinins (BAP, 2iP, kinetin) there was a general trend of increasing shoot number with increasing cytokinin concentration.

• The highest concentration (128gM) of all cytolcinins (BAP, 2iP and kinetin) did not produce the highest number of new shoots.

• For BAP and kinetin the number of new shoots produced by shoots on the highest cytokinin concentration (128tiM) was actually less than the number produced on the lowest concentration (0.5pM).

• BAP was the most successful cytolcinin in terms of the mean number of new shoots produced, at all concentrations.

• At 21.tM concentrations, shoots growing on BAP produced significantly more shoots than 2iP, but not kinetin.

• At 81.1M concentrations, shoots growing on BAP produced significantly more new shoots than shoots growing on both 2iP and kinetin.

• At 321.1M concentrations, shoots growing on BAP produced significantly more shoots that those growing on 2iP, but not kinetin.

• 87% of shoots grew roots on MS + 1g1, 1 AC.

389 Chapter 4. In vitro propagation of Iridaceae species

• Transplanting was initially quite successful, with 76% of plantlets still alive 1 month later.

• Three months after transplanting the percentage of surviving plantlets had decreased to 56%.

• Twelve months after plants were repotted only 26% of those originally transplanted were still alive.

• The majority of plants that died had rotted due to fungal infestation.

• Immature flower buds were also studied as a possible explant source for I. tasmanica in vitro.

• Flower buds were easy to disinfest with 100% of explants remaining sterile 1 month after cultures were initiated.

• Two days after cultures were initiated the majority of buds (81.8%) had opened.

• All of the opened buds turned brown within 21 days in vitro.

• After 1 month in vitro 100% of buds had opened and all were dead.

• There was no regeneration from any of the flower bud explants.

As mentioned previously, seeds are not an ideal source of explants for a micropropagation system. Therefore, other explant sources should be investigated for I. tasmanica. Although flower buds were unsuccessful in the current study, they should not be dismissed as the stage of development used was obviously not optimal for shoot regeneration. A wide range of developmental stages and different media types would be worth trying. Other floral explant types should also be investigated (eg. scape sections and pedicel/peduncle junctions).

In terms of rooting, although roots were produced on MS + 10: 1 AC, it may be worthwhile to try a range of rooting media treatments, which may enhance root development and perhaps increase the chances of survival after transplanting.

Somatic embryogenesis should also be investigated as a possible means of mass propagation of this species.

390 Chapter 5 The Effect of Manipulation of Environmental Parameters on Flowering in Dianella tasmanica

5.1 Introduction

The life history of a is usually characterised by an initial phase of vegetative growth followed by the reproductive phase (Wareing and Phillips, 1981). In perennials (such as the Liliaceae and Iridaceae species studied in this thesis), flowering typically occurs year after year when conditions are appropriate (Kimball, 2004).

The process of flowering involves the conversion of the apical meristem into a floral meristem (Kimball, 2004). The first visible sign that flowering has commenced is when floral initiation (the differentiation of the floral primordia) occurs. The floral primordia develop into , petals, etc. until finally anthesis or pollen-shed is reached (Plummer, 1996).

In some plant species the change from the vegetative to the reproductive phase is determined endogenously and without reference to specific environments but, more often, environmental factors are important and sometimes crucial for the transition to occur (Vince-Prue, 1983). Two of the most important environmental variables that have an effect on flowering are photoperiod (daylength) and temperature (Evans, 1975; Wareing and Phillips, 1981; Weier etal., 1982; Vince-Prue, 1983; Lewis and Warrington, 1985; Rees, 1987, 1992; Villee etal., 1989; Campbell, 1990; Salisbury and Ross, 1992; Le Nard and De Hertogh, 1993c; Pearson et al., 1995; Plummer, 1996; Kimball, 2004).

391 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

It should also be noted that plants have a juvenile phase in which they are unable to respond to environmental stimuli by flowering (Rees, 1987). The concepts of "ripeness to flower" (Klebs, 1918) and the "floral state", i.e. the developmental condition that a plant must achieve before it is able to flower (O'Neill, 1992) recognise this phase.

Therefore, flowering is a complex process as described by Murfet (1977): "Flowering is the end-result of physiological processes, biochemical sequences, and gene action, with the whole system responding to the influence of environmental stimuli and the passage of time."

5.1.1 Effects of photoperiod on flowering The fact that the lifecycles of many plant species are strongly influenced by seasonal changes in daylength was first reported by Garner and Allard (1920). Their pioneering research on the flowering of a mutant tobacco variety "Maryland Mammoth" showed that photoperiod (lengths of daily light and dark periods) was an extremely important factor in the growth and development of plants, particularly in the control of flowering (Wareing and Phillips, 1981; Salisbury and Ross, 1992). Physiological responses to the relative lengths of daylight and darkness (such as flowering) are known as photoperiodism (Vince-Prue, 1983; Rees, 1987; Villee et al., 1989; Campbell, 1990; Thomas and Vince-Prue, 1997).

Plant responses to photoperiod can be categorised into three main groups:

1. Short Day (SD) plants: initially defined as those plants that only flower (or flower earlier) when days are shorter than a critical length (Weier et al., 1982; Vince-Prue, 1983; Villee etal., 1989; Campbell, 1990). However, in the 1940s it was discovered that night length, not daylength, actually controls flowering and other responses to photoperiod (Weier etal., 1982; Campbell, 1990). Therefore, SD plants are more accurately defined as plants that flower when the night length is equal to or greater than a critical length (Villee etal., 1989; Campbell, 1990; Thomas and Vince-Prue, 1997). Some examples of SD plants are listed in Table 5.1. 2. Long Day (LD) plants: initially defined as those plants that only flower (or flower earlier) when the daylength is equal to or longer than a certain critical

392 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

amount (Weier etal., 1982; Vince-Prue, 1983; Villee etal., 1989; Campbell, 1990). However, a more accurate definition is that LD plants flower when the night length is equal to or shorter than the critical length (Villee etal., 1989; Campbell, 1990; Thomas and Vince-Prue, 1997). Some examples of LD plants are listed in Table 5.1. 3. Day Neutral (DN) or indeterminate plants: those plants in which flowering is unaffected by photoperiod (Wareing and Phillips, 1981; Weier etal., 1982; Villee et al., 1989; Campbell, 1990; Thomas and Vince-Prue, 1997). These plants flower in response to some other stimulus - either external (eg. temperature or water stress) or internal (eg. a genetically determined pattern of development) (Weier et a/., 1982; Villee et a/., 1989). Some examples of DN plants are listed in Table 5.1.

Examples of Australian native species that represent the three photoperiodic response types above are listed in Table 5.2.

Photoperiodic responses can be further classified as either obligate (or absolute) or quantitative (or facultative) (Wareing and Phillips, 1981; Weier et al., 1982; Rees, 1987; Salisbury and Ross, 1992; Thomas and Vince-Prue, 1997).

1. Obligate photoperiodic plants: those species that remain permanently vegetative if kept under unfavourable daylength conditions (Wareing and Phillips, 1981). Obligate photoperiodic plants include both SD plants (eg. Xanthium pennsylvanicum) and LD plants (eg. Hyosyamus niger) (Wareing and Phillips, 1981). 2. Quantitative photoperiodic plants: those species that flower earlier under SD or LD, but will ultimately flower even under unfavourable daylength conditions (Wareing and Phillips, 1981). Species that have this response to photoperiod include the SD plants Salvia splendens and rice (Oryza sativa) and the LD plants flax (Linum usitatissimum) and wheat (Triticum) (Wareing and Phillips, 1981).

In addition to the three general response types listed above there are other more complex response types. Some species have dual photoperiodic requirements [known as ambiphotoperiodic-day plants (Thomas and Vince-Prue, 1997)] and will only flower after being exposed to LD followed by SD. Examples of these LSD (Long-Short Day) plants include Bryophyllum and Kalanchoe (Vince-Prue, 1983). Other species require the opposite, SD followed by LD, and are known as SLD plants. For example: Trifolium repens and Echeveria harmsii (Vince-Prue, 1983).

393 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

Table 5.1. Examples of short day (SD), long day (LD) and day neutral (DN) plants.

Genus and species (where known) References 1. Short Day (SD) plants Amaranthus Kigel and Rubin (1985) Begonia boweri Rees (1992) Cattleya amabilis (orchid) Goh and Arditti (1985) Chrysanthemum morifolium Wareing and Phillips (1981), Villee etal. (1989), Campbell (1990) Coleonema aspalathoides (Diosma) Heller et al. (1994). Cosmos bipinnatus "Sensation White" Mattson and Erwin (2005) Rees (1992) Doritis pulcherrima (orchid) Wang et al. (2003) Kalanchoe blossfeldiana Wareing and Phillips (1981), O'Neill (1992) Euphorbia pulcherrima (poinsettia) Villee etal. (1989), Campbell (1990) Pharbitis nil (Japanese Morning Glory) O'Neill (1992) Xanthium strumarium (Cocklebur) Wareing and Phillips (1981), O'Neill (1992)

2. Long Day (LD) plants Allium cepa (onion) Rees (1992) Avena sativa (oat) Wareing and Phillips (1981), Rees (1987) Dianthus superbus (carnation) Wareing and Phillips (1981) Eustoma grandiflorum Grueber et al. (1994), Roh and Lawson (1984), Roh etal. (1989), Islam et al. (2005) Hamelia patens Armitage (1995) Helianthus annuus (sunflower) Goyne et al. (1989), Mattson and Erwin (2005) Iris Campbell (1990) Lactuca sativa (lettuce) Villee etal. (1989), Campbell (1990), Ohio State University (2004) Lysimachia congestiflora Zhang et al. (1995) Raphanus sativus (radish) Wareing and Phillips (1981), Campbell (1990) Salvia farinacea "Strata" Mattson and Erwin (2005)

3. Day Neutral (DN) plants Amaranthus hybridus "Pygmy Torch" Mattson and Erwin (2005) Gladiolus cultivars McKay et al. (1981) Narcissus Rees (1992) ayza sativa (rice) Campbell (1990), Salisbury and Ross (1992) acypetalum caerulea Armitage et al. (1990), Mattson and Erwin (2005) Phaseolus vulgaris (kidney bean) Wareing and Phillips (1981), Salisbury and Ross (1992) Pisum sativum (pea) Campbell (1990), Salisbury and Ross (1992) Thunbergia alata Mattson and Erwin (2005) Tulipa Rees (1992) Zingiber mioga (Myoga) Stirling et al. (2002)

394 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

Table 5.2. Examples of Australian native short day (SD), long day (LD) and day neutral (DN) species.

Species (Family) References 1.Short Day (SD) plants Anigozanthos manglesii () Motum and Goodwin (1987) Chamelaucium uncinatun (Myrtaceae) Shillo etal. (1985), Dawson and King (1993) Lawrencella davenportii () Bunker (1994, 1995) Lawrencella rosea (Asteraceae) Bunker (1994, 1995) Pimelea ciliata (Thymelaceae) Slater et al. (1994)

2. Long Day (LD) plants A nigozanthos flavidus (Haemodoraceae) Motum and Goodwin (1987) Banksia coccinea (Proteaceae) Rieger and Sedgley (1996) Brachycome iberidifolia (Asteraceae) Bunker (1994, 1995) Bracteantha bracteata (Asteraceae) Sharman et al. (1989a,b) Cluysocephalum apiculatum (Asteraceae) Bunker (1994, 1995) craspedioides (Asteraceae) Mott and McComb (1975) Rhodanthe chlorocephala ssp. rosea Sharman et al. (1989b,c) (Asteraceae) Rhodanthe floribunda (Asteraceae) Bunker (1994, 1995) Rodanthe manglesii (Asteraceae) Bunker (1994, 1995) Shoenia cassiniana (Asteraceae) Mott and McComb (1975), Bunker (1994, 1995) Shoenia filifolia ssp. filifolia (Asteraceae) Bunker (1994, 1995) Thoiptomene calycina (Myrtaceae) Beardsell (1991)

3. Day Neutral (DN) plants Anigozanthos flavidus x A. manglesii Motum and Goodwin (1987) (Haemodoraceae) Anigozanthos pulcherrimus (Haemodoraceae) Motum and Goodwin (1987) Anigozanthos rufus (Haemodoraceae) Monnn and Goodwin (1987) Banksia hookeriana (Proteaceae) Rieger and Sedgley (1996) Boronia megastigma () Plummer (1996) Brachycome halophila (Asteraceae) Bunker (1994, 1995) Swainsona formosa (Fabaceae) Williams (1996a)

Intermediate Day (ID) plants, which will only flower between quite narrow daylength limits, also exist (Vince-Prue, 1983; Salisbury and Ross, 1992; Thomas and Vince- Prue, 1997). For example, in a sugar cane cultivar, flowering did not occur unless daylengths were between 12 and 14 hours (Vince-Prue, 1983).

Some species display even more complex responses where their photoperiodic response types change at different temperatures (Rees, 1987; Salisbury and Ross, 1992). For example, Poinsettia (Euphorbia pulcherrima) and Morning Glory (Ipomea purpurea) are SD plants at high temperatures and LD plants at low

395 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica temperatures. Salix (willow) displays the opposite response, with flower initiation occurring under SD at 15°C and LD at 18-24°C (Lavender, 1985). For Gypsophila paniculata cv. "Bristol Fairy" LDs promote flowering, but only at relatively high temperatures. If the night temp is below 12°C the plants remain vegetative even under LDs (Shillo and Halevy, 1982). In the Australian native species Ozothamnus diosmifolius cv. "Cooks Snow White" plants are absolute LD plants when grown in medium-moderate temperatures, at lower temperatures they will flower under both LDs and SDs, while at high temperatures flowering will not occur under either LDs or SDs (Halevy et al., 2001).

Response types of individual species have also been found to vary with differing latitudes (and in turn, differing climates) (Rees, 1987). For example, Themeda australis (kangaroo grass) has a wide distribution throughout and it is a SD plant in the northern tropics, an ID plant in middle-latitude coastal areas, further south the populations are mostly LD plants, while in the dry centre of Australia it is a DN plant, flowering in response to the occasional, seasonally unpredictable rainfall (Evans, 1975).

It should also be noted that daylength also influences not only the transition from vegetative to reproductive growth but also many aspects of flower development (Vince-Prue, 1975). Also, flower development from initiation to anthesis often has different or more stringent requirements than for initiation. For example, floral initiation is under photoperiodic control in coffee plants, but anthesis depends on rain or the temperature drop associated with rain (Rees, 1987). Also, for Zingiber mioga (Myoga) flower initiation is DN, but for flower development LDs are required (Stirling et al., 2002). For the native Australian species Chamelaucium uncinatum (Geraldton Waxflower) floral initiation is controlled by photoperiod while temperature is the major factor affecting flower development (Shillo etal., 1985).

For plants to be able to respond to light they must have a receptor that perceives it (Villee etal., 1989). Phytochrome is the photoreceptor involved in photoperiodism (Vince-Prue, 1983; Wareing and Phillips, 1981; Villee etal., 1989; Campbell, 1990; O'Neill, 1992; Salisbury and Ross, 1992; Weier etal., 1982) and it is perceived in the leaves (especially the young expanding leaves) of the plant (Lang, 1965; Vince-

396 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

• Prue, 1975; Campbell, 1990; Kimball, 2004). The processes that occur in the leaf are known as induction (Vince-Prue, 1983; O'Neill, 1992), while the period between the arrival of the flowering stimulus (sent from the leaves) at the apex and the appearance of flower buds is known as evocation (Vince-Prue, 1983; Lyndon, 1990; O'Neill, 1992).

5.1.2 The effects of temperature on flowering Temperature is another environmental factor that varies throughout the year, and exhibits well-marked seasonal changes, particularly in temperate regions. These seasonal temperature variations also have a profound effect on flowering in many plant species (Wareing and Phillips, 1981). A chilling, or vemalisation, period is required by some species before they will flower (Wareing and Phillips, 1981; Weier et al., 1982; Villee et al., 1989; Campbell, 1990). Under natural conditions this cold period obviously occurs during winter, and often the cold requirement is followed by a LD requirement that prevents flowering from occurring too early in the spring (Weier et al., 1982). Species requiring a vemalisation period include: the Easter lily (Lilium longiflorum) (Rees, 1992), Lilium elegans (Rees, 1992), Chrysanthemum morifolium, primrose (Primula vulgaris), violet (Viola sp.), Sweet William (Dianthus) (Wareing and Phillips, 1981) and many vegetable species, including carrots (Daucus carota) (Villee etal., 1989; Ohio State University, 2004), sugarbeet (Beta vulgaris) (Wareing and Phillips, 1981; Ohio State University, 2004), celery (Apium graveolens) (Wareing and Phillips, 1981), cabbage (Brassica oleracea) (Wareing and Phillips, 1981; Ohio State University, 2004), cauliflower (Brassica oleraceae var. botrytis) (Guo et al., 2004) and other cultivated Brassica species (Wareing and Phillips, 1981). Some native Australian species also require periods of relatively cold temperatures before they will flower, or give the best flowering response after such treatments. For example: for Pimelea ferruginea and P. rosea the best flowering response occurred after plants were exposed to 7 weeks of temperatures below 15°C (King etal., 1992). For Boronia megastigma the number of floral buds initiated and the speed of development were both increased by low night temperatures (Plummer, 1996). The South Australian native Ixodia

397 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica achillaeoides also requires relatively low temperatures for flowering (<17°C) (Weiss and Ohana, 1996). Some native monocots are also induced to flower by low temperatures, including Blandfordia, which requires a chilling period of 4-6 weeks (with temperatures below 10°C) before it will flower (Goodwin and Watt, 1994; Goodwin etal., 1995); while for seasonal flowering clones of Anigozanthos flower initiation is primarily caused by low temperatures (below approximately 15°C) (Wilkins, 1985; Roh and Motum, 1989; Hagiladi, 1983; Goodwin, 1993). As with photoperiod, vernalisation requirements may also be either absolute or facultative. If absolute, flowering will not occur unless plants have been vemalised, while facultative plants will flower sooner if exposed to low temperatures, but will still flower (at a later time) anyway if they are not exposed (Villee etal., 1989). In contrast to species requiring vernalisation, in some cases high temperatures are required for flowering. For example, growth in Freesia corms that do not receive a high temperature treatment is slowed and irregular, while flowering is delayed (Rees, 1992). Flowering is also accelerated by high temperature treatment (30-35°C) in Tulipa, Hyacinthus and Eucrosia (Rees, 1992; Roh etal., 1992).

Temperature appears to be the main factor influencing flowering in the native Australian plants Banksia hookeriana (Sedgley, 1996) and Swainsona formosa (Sturts Desert Pea) (Williams, 1996a), with photoperiod not having any effect, and also in the ornamentals Primula malacoides (Karlsson and Werner, 2002) and a number of Gladiolus cultivars (McKay etal., 1981b).

5.1.3 Photoperiod and temperature interactions As alluded to previously, the effects of photoperiod and temperature should usually be considered in an interactive sense rather than in isolation as plants rarely have a daylength response that is completely independent of temperature (Rees, 1987). For example, flowering is influenced by both photoperiod and temperature in the native Australian species Chamelaucium uncinatum (Shillo et al., 1985), Thryptomene calycina (Beardsell, 1991), Lechenaultia formosa and L. biloba (Worrall, 1996b). Also, as mentioned previously, many plants have different photoperiodic responses at different temperatures (see section 5.1.1).

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5.1.4 Manipulation of environmental parameters: flower "forcing"

Once the environmental factors (eg. temperature and photoperiod) that trigger flowering are known it is possible to use this information to "force" plants to flower out of season (Lewis and Warrington, 1985; Rees, 1987; Campbell, 1990; Dewi, 1993; Le Nard and De Hertogh, 1993c). Flowering can be forced to occur earlier than normal or it can be delayed to target specific markets (Le Nard and De Hertogh, 1993c). For example, in the USA Chrysanthemums are SD plants that normally flower in Autumn. However, by punctuating each long night with a flash of light (effectively turning one long night into two short nights) flowering can be delayed until Mothers Day (Campbell, 1990). Forcing of flower bulbs, in particular, as cut flowers or potted plants has been widely practiced (Le Nard and De Hertogh, 1993c) since as early as the 1920s (Salisbury and Ross, 1992). However, it is only in relatively recent times that studies into the manipulation of flowering of Australian native species have taken place. Only species with commercial importance have been studied in detail, such as Chamelaucium uncinatum (Shillo et al., 1985; Dawson and King, 1993), Lechenaultia (Worrall, 1996b), Swainsona formosa (Williams, 1996a), Banksia (Rieger and Sedgley, 1996; Sedgley, 1996), Acacia (Sedgley, 1985, 1986), Pimelea (King etal., 1992; Slater etal., 1994), Boronia (Lamont, 1985a,b; Richards, 1985; Roberts and Menary, 1989a,b, 1990, 1994; Day, 1993; Day etal., 1994; Bussell, 1995); native daisies (Asteraceae family) (Mott and McComb, 1975; Sharman etal., 1989a,b,c; Bunker, 1994, 1995), Thryptomene calycina (Beardsell, 1991), Ixodia achillaeoides (Weiss and Ohana, 1996) and Ozothamnus diosmifolius (Halevy etal., 2001). In terms of native monocots, only Anigozanthos (Wilkins, 1985; Motum and Goodwin, 1987; Roh and Motum, 1989; Hagiladi, 1983; Goodwin, 1993) and Blandfordia (Goodwin and Watt, 1994; Goodwin etal., 1995) appear to have been studied in detail.

Preliminary experimentation with the native Tasmanian Liliaceae species Dianella tasmanica suggested that flowering was mainly controlled by photoperiod, with floral initiation occurring under SDs (Sward, 1995). The development and elongation of the floral stem was promoted by relatively high temperatures (Sward,

399 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

1995). By growing this species at a constant temperature of 20°C with an 8 hr photoperiod it was possible to force plants to flower at least 3 months earlier and with a higher percentage than the control (Ibid.).

5.1.5 Smoke treatments A smoke treatment is sometimes used by commercial producers of ornamental flower bulb species to promote early flowering. Leaves, rice straw or other plant materials are burnt to produce the smoke (Imanishi and Fortanier, 1983). Such treatments have been common practice in Japan for over 50 years (Imanishi and Fortanier, 1983; Rees, 1992) and have resulted in many desirable effects including increased flowering percentages, earlier floral initiation, faster flower development (Rees, 1992) and a more uniform flowering time which facilitates flower cutting (Swart and Schipper, 1982). Species that have their flowering promoted by smoke include: Narcissus tazetta (Imanishi, 1983, 1996; Imanishi and Fortanier, 1983; Imanishi et al., 1986; Chang and Yang, 1987; Rees, 1985, 1992; Hanks, 1993), Dutch Iris (Schipper, 1982,1984; Swart and Schipper, 1982; Imanishi and Fortanier, 1983; Imanishi et al., 1986; Rees, 1992; Imanishi, 1996) and Freesia (Imanishi and Fortanier, 1983; Uyemura and Imanishi, 1983; Rees, 1992; Imanishi, 1978, 1993). Smoke treatments have also produced other desirable effects in some species including: a decrease in flower bud "blasting" and of the occurrence of three-leaved plants (which will not flower) in the Iris x hollandica cultivar "Ideal" (Schipper, 1982); and the formation of flowers by bulbs of sub-critical size (i.e. bulbs that are too small to flower under normal conditions) in Iris (Schipper, 1982) and Narcissus tazetta (Hanks, 1993). Smoke treatments also terminated dormancy sooner in Freesia corms, which led to earlier flowering (Hayashi, 1974). Smoke treatments are also cost effective as they replace the need for high temperature treatments, which were the standard treatment to promote flowering for many years (Schipper, 1984; Rees, 1992).

In order to simplify smoke treatments, Uyemura and Imanishi (1983) tried to identify the effective components within smoke. They discovered a marked increase in the content of three hydrocarbons - ethane, ethylene and propylene, that coincided with

400 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica the active generation of smoke. Carbon monoxide and a 2-12 fold increase in CO 2 and a 0.3-1% decrease in 02 (when compared to normal concentrations in air) were also detected in the smoke (Uyemura and Imanishi, 1983). After a series of experiments to screen the possible effective components of smoke they concluded that ethylene was the only active component (Uyemura and Imanishi, 1983; Imanishi, 1996). Many other authors have indeed reported that ethylene produces similar effects to those caused by smoke treatments (eg. Stuart etal., 1966; Hayashi, 1974; Durieux and Kamerbeek, 1974; Kamerbeek and De Munk, 1976; Masuda and Asahira, 1980; Swart and Schipper, 1982; Imanishi and Fortanier, 1983; Uyemura and Imanishi, 1983; Imanishi etal., 1986; Imanishi and Yue, 1987; Rees, 1992; De Munk and Schipper, 1993; Le Nard and De Hertogh, 1993c; Hanks, 1993; Imanishi, 1996; Whitehead, 1996; Botha etal., 1998). As well as promoting flowering in the same species previously listed as responding to smoke treatments, ethylene also promotes flowering in Ornithogalum thyrsoides and breaks dormancy in the corms of Freesia and Gladiolus (Le Nard and De Hertogh, 1993c). Only small quantities of ethylene are required to produce the desired effects (Rees, 1992). The recommended concentration of ethylene for storage rooms is initially 500WL due to uptake of the gas or to leakage from the storage room (De Munk and Schipper, 1993). Exposure of bulbs to ethylene is recognised as being more effective than heat treatments and it is recommended that bulbs are exposed to ethylene for 24 hr or are dipped for 15 min in a solution containing ethephon (Le Nard, 1983; Cascante etal., 1989). However, the necessary concentrations and exposure times vary according to species and even cultivar.

"Burning-over", a process whereby bulbs that are not lifted following the growing season have their winter straw cover burnt-over to reduce weed pressure, produces a higher percentage of flowering Iris bulbs than those that have not been treated in this manner (Price, 1971). Several Narcissus cultivars have also flowered earlier following burning-over (Wolfe and Horton, 1958). However, the promotion of flowering has been attributed to the ethylene in the smoke, rather than the warm temperatures of the bare, burnt soil (Tompsett, 1985b).

401 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

Many native Australian monocots also respond to fire by flowering (Table 5.3), and in the majority of cases it is only following fire that they will flower abundantly (Gill, 1981a) (Plate 5.1).

Table 5.3. Australian species that flower profusely following fire. Source: Adapted from Gill (1981a) and updated.

Family Species Reference Cyperaceae Cyathacha eta tadiandra Ingwersen (1977) Doryanthaceae Doryanthes excelsa Nash (1996), Stewart (1999a), Denham and Auld (1999, 2002) Haemodoraceae Anigozanthos pukherrimus Lamont and Runciman (1993) paniculatum Baird (1977) Haemodorum spicatum Baird (1977) Macropidia fuliginosa Lamont and Runciman (1993) Iridaceae Diplarrena moraea Conn (1994) Liliaceae Blandfordia nobilis Johnson etal. (1994), Johnson (1996a), Stewart (1999a) Blandfordia punicea Personal observation Burchardia umbellata Baird (1977) Orchidaceae Burnettia sp. Barnett (1984), Bates (1984) Caladenia unita Erickson (1965) Caladenia menziesii Willis (1962) Caladenia sp. Baird (1984), Barnett (1984), Bates (1984) Diuris sp. Baird (1984), Barnett (1984), Bates (1984) Eriochilus scaber Erickson (1965) Glossodia sp. Baird (1984), Barnett (1984), Bates (1984) Leptoceras fimbriata Erickson (1965) Lyperanthus nigricans Willis (1962), Erickson (1965) Lyperanthus sp. Baird (1984), Barnett (1984), Bates (1984) Prasophyllum sp. Baird (1984), Barnett (1984), Bates (1984) Xanthorrhoeaceae australis Gill and Ingwersen (1976), Gill (1981a), Curtis (1998) Xanthorrhoea australis (syn. Specht et al. (1958) X semiplana) Xanthorrhoea fulva Taylor et al. (1995, 1998), McFarland (1990) Xanthorrhoea glauca J. Taylor, personal observation Xanthorrhoeajohnstonii Billow-Olsen et al. (1982) Xanthorrhoea latifolia J. Taylor, personal observation Xanthorrhoea macronema Rogers (1975) Xanthorrhoea minor Gill (1981a) X preissii Baird (1977), Lamont and Downes (1979), Lamont et al. (2000) Xanthorrhoea tateana J.I. Wooley, pers.comm.

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Plate 5.1. Blandfordia sp. flowering prolifically 10 months after the passage of an intense fire in the Sydney region. Source: Stewart (1999a).

These species usually survive fires as their meristematic regions are protected underground in the form of rhizomes, corms or buds (Denham and Auld, 1999, 2002). Following the passage of a fire they are able to resprout, flower and then release seeds (Gill, 1981a; Frost, 1984; Johnson etal., 1994; Bond and Van Wilgen, 1996; Carrington, 1999). Species like these which are generally dependant on post- fire flowering for recruitment are known as "pyrogenic flowering resprouters" (Denham and Auld, 2002).

403 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

Perhaps the most well known pyrogenic flowering resprouters are the grasstrees from the Xanthorrhoea genus. This genus has indeed been the most researched (Table 5.3). To ascertain the causes of the flowering response to fire, Gill and Ingwersen (1976) studied Xanthorrhoea australis. Plants were exposed to one of four treatments: burning, clipping of crowns, ethylene treatment or control (no treatment). Their results suggested that inflorescence production was influenced by at least three stimuli: a seasonal trigger (such as photoperiod), leaf removal and smoke (especially the ethylene content), with the strongest stimuli being the removal of the foliage (Gill, 1981a). Studies on X fulva showed that burning produced more than clipping (Taylor etal., 1998) and the season of both types of disturbance was significant. They concluded that floral induction in X fulva must be a combination of a seasonal trigger, crown removal, between-fire interval and some other factor(s) - perhaps smoke as proposed by Gill (1981a) (Taylor etal., 1998). The importance of the role of smoke in stimulating flowering has also been discussed by Curtis (1998) for X australis and Lamont etal. (2000) for X preissii.

The causes of the flowering response for the species mentioned in Table 5.3 are likely to vary (Gill, 1981a). However, possible reasons for fire-enhanced flowering may be the increased soil moisture (Kauffman etal., 1997), light and soil nutrients (Raison, 1979, 1980; Humphreys and Craig, 1981; Kauffman et al., 1997) that occur in the post-fire environment. Decreased competition or diurnal temperature changes may also be involved (Martin, 1966). Regardless of the reasons, the responses have probably evolved due to selection for increased seed production following fire (Gill, 1981a), which could be related to various environmental pressures. For example, the chances of pollination and outcrossing would be increased during a mass flowering event, as would the chances of satiating seed predators (Gill, 1981b; O'Doud and Gill, 1984; Brewer and Platt, 1994).

Flowering responses to fire are not limited to Australian species. Indeed, such responses have also been reported for Hypoxis sessilis and Hypoxis wrightii (Herndon, 1988) and the wet prairie grass species Muhlenbergia capillaris (Main and Barry, 2002; Snyder, 2003), Paspalum monostachyum and Schizachyrium rhizomatum (Main and Barry, 2002) in South Florida, USA; Californian chapparal

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Liliaceae species including Brodiaea ixioides (Stone, 1951), Dichelostemma cap itatum (Anderson and Roberts, 2000) and Zigadenus fremontii (Tyler and Borchert, 2003); and Haemanthus canaliculatus (Amaryllidaceae) from South Africa (Levyns, 1966).

Preliminary experimentation with the native Tasmanian Liliaceae species Dianella tasmanica suggested that a smoke treatment may increase the percentage of flowering plants, with 26.7% of smoke treated plants flowering, compared to only 7.1% of control plants (Sward, 1995). Smoke treated plants also flowered earlier than the control plants (Ibid.).

5.1.6 Aims of the current study Due to the horticultural potential of the Dianella genus it was considered worthwhile to further investigate the effects of temperature and photoperiod on flowering in this species. Initial experimentation had shown that flowers were initiated under short photoperiods (during winter), and that the elongation of the floral stem was triggered by warmer temperatures (during spring). Therefore, the aim of this experiment was to determine whether D. tasmanica plants kept in cold storage with a short photoperiod (thus mimicking winter conditions) could be forced to flower by being given a "heat shock" - placing them under warmer conditions (thus mimicking spring/summer conditions which is when they flower naturally). If this was successful, it would be possible to store plants under cold conditions and then force them to flower on demand by placing them under warmer temperatures; thus, allowing year-round production of flowering plants.

In light of the variety of Australian native monocot species that flower abundantly following fire (particularly Blandfordia nobilis, which is also a member of the Liliaceae family) and the initial results suggesting that D. tasmanica may have a flowering response to smoke (Sward, 1995), it was thought prudent to further investigate this response. Thereby determining the possibility of forcing D. tasmanica using a smoke treatment or other fire-related treatments including foliage removal, burning and the application of burnt soil.

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5.2 Materials and Methods

5.2.1 The effect of cold storage followed by a heat shock on the flowering of Dianella tasmanica

Sixty D. tasmanica plants of similar size were selected for this experiment. All of these plants were originally collected from Geeveston, southern Tasmania, and had been grown in pots (in native potting mix, see Appendix 3.1) for approximately 1.5 - 2 years (being divided and repotted when required).

One month prior to the experiment none of the plants were showing any sign of flowering. However, to ensure that floral initiation had not occurred yet, 6 other plants (from the same location and of similar size, but not to be used in the experiment) were studied more closely. A shoot from the end of the rhizome was selected from each plant for dissection to determine whether floral initiation had already occurred. Terminal shoots were selected as it had previously been determined that floral stems were terminal on the rhizome (Sward, 1995). Leaves were carefully removed from the base of the shoots to expose the apical meristems which were then removed with the aid of a dissecting microscope. Longitudinal sections of the meristem were taken using a freeze microtome. No floral initials were observed on any of the meristems, which still appeared to be in the vegetative state. Therefore, the experiment could be continued.

The 60 plants were placed into two growth cabinets both set at 10°C with an 8 hr photoperiod (cold temperature, short photoperiod) to mimic winter conditions, when the plants would not be flowering naturally. They remained under these conditions for approximately 7 months (starting in late August, when plants would not naturally be flowering). All plants were checked for signs of flowering on a weekly basis.

Following the 7 month period at 10°C and 8 hr photoperiod the remaining plants (some had died) were placed in growth cabinets under different environmental conditions (Table 5.4). In all growth cabinets the relative humidity was approximately 65%. They remained in the new treatments for approximately 8

406 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

months, during which time they were checked weekly for signs of flowering, as before.

Table 5.4. The environmental conditions (temperature and photoperiod) that D. tasmanica plants were grown in following an initial 12 months at 10°C with an 8 hr photoperiod. p.p = photoperiod; temp = temperature.

Treatment Description Number of plants 1.Control 10°C, 8 hr p.p 12 2. Heat shock, short p.p 25°C, 8 hr p.p 12 3. Heat shock, long p.p 25°C, 16 hr p.p 12 4. Cold temp, long p.p 10°C, 16 hr p.p 12

It was hoped that the warmer conditions in treatments 2 and 3 (Table 5.4) would "shock" the plants into flowering, as this temperature would be similar to the spring/summer conditions that Dianella naturally flowers during. By also including long and short photoperiods associated with both the cold and warm temperatures it would be possible to determine whether the temperature alone was producing any effects, or whether the photoperiod was also involved. As a control the final quarter of plants remained under the initial conditions (Table 5.4) for the duration of the experiment. The final flowering results were analysed using Pearson's Chi-Squared statistic, and pairwise comparisons.

5.2.2 The effect of smoke, burning and foliage removal treatments on flowering in Dianella tasmanica

The aim of this experiment was to determine the effects of various treatments associated with fire on flowering in Dianella tasmanica. To ensure that the treatments and their effects were similar to those that would occur in a natural bushfire, the plants to be studied were planted in an area of natural bushland (on private property) where D. tasmanica would naturally grow.

An area of dry sclerophyll vegetation at Kettering, southern Tasmania, was selected for use in this experiment. Natural vegetation growing on the site included

407 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

Eucalyptus, Lomandra, Diplarrena, Arthropodium and Epacris. The area was slashed to remove the smaller vegetation, with larger trees remaining.

Five treatments each containing 5 replicates of 8 plants were to be used. Therefore, the experimental area was measured out to incorporate 25 plots, each 1 m wide x 0.5 m breadth. The dimensions of the experimental area are shown in Fig. 5.1.

19.5 m 0.5 m 1.5 m 17.4m 4.5 m 12.6m

Figure 5.1. Dimensions of the experimental area for the D. tasmanica smoke, burning and foliage removal experiment. There were 10 plots across the top row, 9 across the middle row and 6 across the bottom row.

Two hundred plants of a similar size were selected for use in this experiment. All were originally collected from Geeveston, southern Tasmania, and had been growing in pots (in native potting mix) for approximately 1.5 - 2 years (being divided and repotted when required). The plants were randomly allocated to the 25 plots and planted. They were left for 2 weeks to establish before any treatments were applied (Plate 5.2).

The treatments to be applied were randomly allocated to each experimental plot (Fig. 5.2). Experimental treatments are described in Table 5.5.

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Chapter 5. The effect of manipulation of environmental parameters °glowering in D. tasmanica

Plate 5.2. Dianella tasmanica experimental plots for the smoke and flowering experiment (prior to application of treatments).

FR BS FR

FR BS FR BS

FR BS BS

Figure 5.2. The allocation of treatments to each experimental plot for the D. tasmanica smoke, burning and foliage removal experiment. C = control; S = smoke; B = burn; FR = foliage removal; and BS = burnt soil.

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Table 5.5. Description of each treatment for the D. tasmanica smoke, burning and foliage removal experiment.

Treatment Description 1. Control No treatment 2. Smoke Damp leaf litter (collected from the surrounding area of native vegetation) was burnt in an apiarists' smoker. The smoke produced was puffed onto the plants which were then covered with garbage bags to contain the smoke. The garbage bags were left covering the plants for 30 min. See plate 5.3a. 3. Burn Leaf litter (collected from the surrounding area) was placed on top of the plants and some newspaper was pushed underneath. The newspaper was lit and the fire remained alight until it burnt out naturally (approximately 15-20 min). See Plate 5.3b. 4. Foliage The leaves were removed from the plants using secateurs, removal so that only the base of the shoots remained (approximately 5 cm long) See Plate 5.3c. 5. Burnt soil Some soil from the area surrounding the experimental plot was collected and placed in a pile. The pile was covered with leaf litter and a small amount of newspaper. This was lit and the soil was burnt for approximately 30 min. The soil was left to cool before being placed around the base of the plants in this treatment. See Plate 5.3d.

The pH of the soil was taken from 4 different plots, prior to any treatments being performed. Two weeks after the treatments were applied, the pH was measured again. The foliage removal treatment was performed first, followed by the burn treatment. Prior to the burn treatment all other plots were covered using large garbage bags, which were weighed down around the edges with soil and rocks, ensuring that there weren't any gaps. The soil for the burnt soil treatment was burnt at the same time as the burn treatment was being done. The smoke treatment was done next, with all other plots remaining covered with the garbage bags. After all of the treatments had been completed, the garbage bags were removed from the plots, and the burnt soil (which had now cooled) was placed around the base of the plants allocated to this treatment.

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Plate 5.3. Application of treatments to Dianella tasmanica plants. a = smoke, b = burn, c = foliage removal, and d = burnt soil.

The plants were checked weekly for flowering, and other relevant observations were made, including the lengths of the flowering stems, any abnormalities in the flowers/flower stems, and re-growth from the foliage removal and burn treated plants.

As the mean and total number of plants that developed flowering stems was quite low, the data could not be analysed by ANOVA. Instead, the percentage data was analysed using Pearson's Chi-Squared statistic.

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5.3 Results

5.3.1 The effect of cold storage followed by a heat shock on the flowering of Dianella tasmanica

During the initial cold storage, short photoperiod treatment (10°C, 8hr) 16.7% of plants flowered. Flowering stems were produced by 5% of plants after nearly two months under these conditions. By mid-September (approximately 2.5 months after placement in the growth cabinets) 15% of plants had produced flowering stems. At this time, there were plants with quite long flowering stems outside, under natural temperature and photoperiod conditions. By mid-late September 16.7% of plants had flowering stems. It took until early December (approximately 3 months after flowering stems had begun to show) for half of the plants with flowers to reach anthesis. Five plants (8.3%) died during the initial cold storage treatment.

At the end of the experiment (after 8 months in the second treatment) Pearson's Chi- Squared statistic showed that there were differences between treatments in respect of the percentage of plants that flowered (x 2 = 14.4; p = 0.00). The highest percentage of flowering plants occurred in the 25°C, 8 hr treatment, where 50% of plants flowered (Fig. 5.3) (Plate 5.4). This result was significantly greater than the percentage of flowering plants from both 10°C treatments (8 hr and 16 hr) (p=0.00), in which no flowering was observed at all (Fig. 5.3; Table 5.6). The only other treatment in which plants flowered was the 25°C, 16 hr treatment. Only a low percentage of plants flowered (16.7%), and this result was not significantly different to any of the other treatments (Fig. 5.3; Table 5.6).

In terms of time to flowering, the majority of plants in the 25°C, 8 hr treatment had flowering stems after only one month in these conditions. Those from the 25°C, 16 hr treatment took slightly longer, with floral stems observed after approximately 2 months. In both of these treatments the time from the beginning of elongation of the floral stem to anthesis was approximately 3.5 weeks.

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Figure 5.3. The percentage of D. tasmanica plants that flowered in each combination of the temperature and photoperiod treatments.

Plate 5.4. D. tasmanica flowering plant from the 25°C, 8 hr treatment. Scale bar = 5 cm.

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Table 5.6. p-values for pairwise comparisons of percentage flowering for D. tasmanica plants from various temperature and photoperiod treatments using Chi-Squared tests. Significant values (p < 0.05) are highlighted.

p-values Treatment 2 3 4 1 0.14 1.00 2 0.08 3 0.14

Ti = 10°C, 8 hr T3 = 25°C, 16 hr

T2 = 25°C, 8 hr T4 = 10°C, 16 hr

By the end of the experiment 4 plants had died. Half of these were from the 25°C, 8 hr treatment and the other half from the 25°C, 16 hr treatment. One dead plant from each of these treatments had flowered prior to dying.

5.3.2 The effect of smoke, burning and foliage removal treatments on flowering in Dianella tasmanica

5.3.2.1 Flowering The first plants to produce flower stems were from the smoke, foliage removal and burnt soil treatments. These plants had stems 29 days after the treatments were applied, with the most advanced stem being 16 cm long (from the foliage removal treatment) (Table 5.7), which suggested that this plant may have been the first to initiate the flowering process. The highest percentage of plants with flowering stems occurred in the smoke treatment at this stage (Table 5.7). Although the smoke, foliage removal and burnt soil treatments had a higher percentage of flowering plants than the other treatments, the differences were not significant (x2 = 6.2, p=0.18).

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Table 5.7. The percentage of D. tasmanica plants with flowering stems 29 days after various smoke, burning and foliage removal treatments were applied.

Treatment Percentage of Length of stems (cm) plants with flowering stems 1. Control 0 N/A 2. Smoke 7.5 10, 7, 5 3. Bum 0 N/A 4. Foliage removal 2.5 16 5. Burnt soil 2.5 6

Plants from the control and burn treatments did not have noticeable flowering stems until 42 days after those from the other treatments (71 DAA, "days after application") and the percentages were quite low (Table 5.8). By this time, 12.5% and 10% of the smoke and foliage removal treatments, respectively, had flowering stems. There was no change in the number of plants with flowering stems from the burnt soil treatment (Table 5.8).

Table 5.8. The percentage of D. tasmanica plants with flowering stems 71 days after various smoke, burning and foliage removal treatments were applied. * = tops of stems have been eaten and therefore could not be measured.

Treatment Percentage of Length of stems (cm) plants with flowering stems 1. Control 2.5 5 2. Smoke 12.5 20, 15, 9 3. Bum 5.0 * 4. Foliage removal 10.0 25 5. Burnt soil 2.5 10

The percentage of plants with flowering stems did not change beyond 71 DAA for any of the treatments, even though results were collected until 168 DAA. For the burnt soil treatment, the results did not change from the first time data was collected (29 DAA). Although the smoke and foliage removal treatments had a higher

415 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica percentage of flowering plants than the other treatments, the differences were not significant (x2 = 5.4, p=0.25).

Flowers did not reach anthesis until 99 DAA, with the first plants with open flowers being: one from the smoke treatment (with a flowering stem 38.5 cm long) (Plate 5.5a) and the only one with a flowering stem from the control treatment (flowering stem only 13 cm long) (Plate 5.5b). It is possible that other plants may have had open flowers at this time, but as a large proportion of the flowering stems had their tops eaten by wildlife, this could not be determined. One deformed flowering stem was also noted on a plant from the smoke treatment (Plate 5.6).

Plate 5.5. Flowering D. tasmanica plants from the smoke (a) and control (b) treatments. Note the normal height of the stem on the smoke treated plant (38.5 cm) and the unusually short stem on the control plant (13 cm).

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Plate 5.6. Deformed flowering stem on a D. tasmanica plant from the smoke treatment.

5.3.2.2 Regrowth from defoliation treatments Regrowth was initially noted 71 DAA from a burn treated plant, which had one new shoot starting to grow. However, the majority of regrowth did not occur until 168 DAA, at which time there was regrowth from 4 non-flowering plants in both the burn and foliage removal treatments. Regrowth from a burnt plot is shown in Plate 5.7.

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Plate 5.7. Regrowth from D. tasmanica burn treated plot. Note the burnt stems in the centre (a) and the new growth (b).

5.3.2.3 Soil pH The soil in the experimental area was initially quite acidic, with a pH of 5.5 obtained from 4 different plots. However, two weeks after the burning treatments, the top layer of the soil (including the ash) was highly alkaline, with a reading of approximately 9. Lower down in the soil below the ash layer the pH was approximately 6. It should be noted that the second time the pH was taken it was a very rainy day and the soil was quite wet. The pH of the soil in all other treated plots had not changed since the initial readings.

418 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

5.4 Discussion

5.4.1 The effect of cold storage followed by a heat shock on the flowering of Dianella tasmanica

Preliminary investigations into the flowering behaviour of D. tasmanica suggested that this species is a short day plant and that the development and elongation of the floral stem is promoted by relatively high temperatures (Sward, 1995). This follow- up experiment supported this theory, with the highest percentage of flowering plants occurring in the short day (8 hr), high temperature (25°C) treatment.

Plants were initially placed in a cold storage, short photoperiod (10°C, 8 hr) treatment in the hope that they would initiate flowering, but that the development and elongation of the floral stem would be prevented until they were placed under warmer conditions. Thereby allowing the manipulation of flowering so that plants could be forced to flower on demand. However, during this initial period a small percentage of plants (16.7%) flowered. It is most likely that floral initiation had already occurred in these plants prior to the commencement of the experiment and that the 10°C temperature was not cold enough to prevent the elongation of the floral stem. This idea is supported by the fact that in a previous experiment there was no flowering observed in any plants grown at the lower temperature of 5°C under both short and long photoperiods (Sward, 1995). Also, the previous experiment began in early winter when it was less likely that floral initiation had occurred, rather than the late winter start in this experiment. Although a number of plants that had previously been grown under the same conditions as the experimental plants were checked for floral initiation by dissection and observation of the apical meristem and found to still be vegetative, it is possible that initiation had taken place but that no visible changes had occurred yet; i.e. plants were at the stage of evocation (Lyndon, 1990). It did take approximately 2.5 months for the majority of plants in the cold storage conditions to produce flowering stems which suggests that this could indeed be the case. The fact that there were plants with flowering stems outside, under natural

419 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

conditions, at the same time as those in the growth cabinets also supports the theory that initiation may already have taken place prior to the experiment.

Under the initial cold storage period, it took approximately 3 months from the time flowering stems were first observed until anthesis, which was very slow compared to the 3 weeks it took for plants in the 25°C, 8 hr treatment. As plants from both treatments were in the same short photoperiod this difference is obviously due to the temperature, with flowering stems developing much faster in the warm 25°C temperature. It is well-known that higher temperatures generally accelerate anthesis when plants are grown in inductive photoperiods (Rees, 1987). In the previous experiment with D. tasmanica a three week time from first observation of the floral stem to anthesis was also observed at 20°C (Sward, 1995).

A small percentage of plants died in both the initial cold storage treatment and then in the 25°C, 8 hr and 25°C, 16 hr treatments. This is perhaps due to the cold temperatures in the first case, but could also be due to insufficient water, which is most likely the case in the 25°C treatments, where the plants may have dried out.

The highest percentage of flowering plants (50%) occurred in the 25°C, 8 hr treatment which reiterates the theory that D. tasmanica is a SD plant that requires a warm temperature for development of the floral stem (Sward, 1995). Another native monocot Anigozanthos manglesii is also induced to flower by SDs (Motum and Goodwin, 1987). Like in D. tasmanica, temperature also has a strong influence on the rate of floral development in Anigozanthos species, with the rate of floral evocation increasing as the temperature increases to 27°C (Motum and Goodwin, 1987). The Geraldton Waxflower, Chamelaucium uncinatum, also responds similarly to D. tasmanica in that photoperiod (SD) is the main factor affecting floral initiation, while flower development is mainly affected by temperature (Shillo et al., 1985). As a relatively small (but not statistically different) percentage of plants also flowered in the 25°C, 16 hr treatment this suggests either that the temperature has a more important role than initially thought or that at the higher temperature the photoperiod has less influence over flowering. The photoperiodic response type often does change at different temperatures for some species (Rees, 1987; Salisbury and Ross, 1992). For example, LDs promote flowering of Gypsophila paniculata

420 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica cultivar "Bristol Fairy", but the long photoperiod is only effective at relatively high temperatures. At night temperatures below 12°C plants remain vegetative even in LDs (Shillo and Halevy, 1982). Also, for the Australian native Ozothamnus diosmifolius cultivar "Cooks Snow White" plants are absolute LD plants when grown in medium - moderate temperatures, at lower temperatures they will flower under both LDs and SDs, while at high temperatures flowering will not occur under either daylength (Halevy et al., 2001). As there were no flowering plants observed in the other LD treatment, at the colder 10°C temperature, this suggests that it is the temperature eliciting the response rather than the photoperiod. In the preliminary flowering experiment there was no flowering observed in the long day, high temperature treatment (Sward, 1995). However, the temperature used in that experiment was slightly colder (20°C), which may not have been warm enough to over-ride the photoperiodic control. As was the case for the preliminary experiment, no flowering was observed in the second 10°C, 8 hr treatment, which suggests that these conditions are not conducive for floral initiation. Although some plants did flower in this treatment initially this was most likely because these plants had already initiated flowering prior to the start of the experiment. The fact that no further flowering was observed in this treatment also supports this idea. Therefore, it is possible that there are photoperiod and temperature interactions occurring for D. tasmanica, whereby at a relatively high temperature the plants may be facultative SD plants, which will flower earlier and in higher numbers under SDs, but will still flower under LDs.

Floral stems were observed on plants in the 25°C, 8 hr treatment quite quickly (after only 1 month in these conditions), which means that it would be possible to force plants to flower quite quickly when required. It would also be possible to program the production of flowering plants by keeping them under cold temperatures with a short photoperiod and then transferring them to warmer conditions two months before they were required (as it takes a further 3 weeks until anthesis).

It is important to be aware that before a plant can flower in response to its environment (particularly photoperiod and temperature), the leaves that detect the environmental change must have reached the condition known as "ripeness to

421 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica respond" and meristems must be competent to respond to the stimulus from the leaves (Salisbury and Ross, 1992). Therefore, it is possible that, as the ages of the plants used in this experiment were not known and all plants were not of the exact same size, some of the plants may not have been competent to flower. Many mono cots need to be of a certain size or have a specific number of leaves before they are able to flower, even under favourable environmental conditions. For example, Anigozanthos rhizomes need to be of a certain size before floral initiation is assured (Motum and Goodwin, 1987), many flower bulb species must reach a certain size before they are able to flower (Schipper, 1982) and the Iris x hollandica cultivar "Ideal" will not flower if it only has three leaves (or less) (Schipper, 1982).

In conclusion, this experiment has shown that D. tasmanica plants can be held at a cold temperature (10°C) with a short (8 hr) photoperiod for up to seven months and then forced to flower by placing them into a warmer 25°C temperature with the same short photoperiod. However, the long term effects of keeping this species at such a cold temperature must be further investigated as the shoot growth and overall health of the plants would probably be retarded under these conditions. In a previous experiment the healthiest plants, in terms of number of new shoots produced and leaf colour and appearance, occurred under a combination of long days and high temperature (20°C) (Sward, 1995). In the previous experiment the long day, high temperature plants did not flower, while at the slightly warmer (25°C) temperature in the current experiment a small percentage did. Therefore, it may be possible to choose healthy stock plants that could be maintained in a vegetative state under long days at high temperature (20°C), during which time new shoots would be formed. When plants were required for flowering it would perhaps be possible to divide new shoots (which were competent) from the stock plant and place these under the warmer 25°C temperature with a short photoperiod to induce flowering. A system such as this would make it possible to achieve year-round flowering and thus increase the range of markets available throughout the year.

422 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

5.4.2 The effect of smoke, burning and foliage removal• treatments on flowering in Dianella tasmanica

Preliminary investigations suggested that D. tasmanica exhibits a similar flowering response to fire (particularly to smoke) as other native Australian monocots (Sward, 1995). This follow-up experiment, incorporating other aspects related to fire, supports this suggestion with the highest percentage of flowering plants occurring in the smoke treatment.

The first plants to produce flowering stems in the current experiment were from the smoke, foliage removal and burnt soil treatments. These plants had stems approximately 1.5 months earlier than those from the control and bum treatments. Therefore, these three treatments, especially the smoke treatment (which had the highest percentage of plants with flower stems at this stage), induce flowering earlier than the control. The preliminary D. tasmanica experiment also showed that plants treated with smoke flower earlier, but only two weeks prior to the control plants in that case (Sward, 1995). Some ornamental flower bulb and corm species also flower earlier when treated with smoke (Rees, 1992).

The fact that the burnt plants began to flower later than the plants from the other treatments (except for the control) is perhaps due to the harshness of this treatment. Physically burning the plants would probably cause more damage than, for example, the foliage removal treatment, as when the foliage was removed care was taken not to cut too low so that the apical meristem would not be damaged. However, during the burn treatment there was no control over how low the fire burnt to on the shoot bases, and the meristem was probably affected by the heat. Therefore, burnt plants would probably take longer to recover and be physically able to flower.

This experiment was monitored until 168 DAA. However, the percentage of plants with flowering stems did not change beyond 71 DAA for any of the treatments, which shows that the response to these fire-related treatments was quite rapid. This rapid flowering response to fire and subsequent seed release is common amongst other pyrogenic flowering resprouters (i.e. species that are dependant on post-fire flowering for recruitment) (Denham and Auld, 2002). However, the response was

423 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica considerably faster than that noted for another native Australian monocot species, Dolyanthes excelsa, which did not flower until 19 months after a fire (Denham and Auld, 1999, 2002). However, this species is a large arborescent monocot (Smith, 2000) and is therefore morphologically quite different to D. tasmanica.

In the current experiment the smoke treatment produced the highest percentage of flowering plants (12.5%), which was five times higher than the percentage of flowering control plants. Although these results were not significantly different, it is still a very positive result considering that the plants were only exposed to the smoke for 30 min. Traditionally for flower forcing of Narcissus tazetta the bulbs are exposed to smoke for several hours on ten consecutive days (Imanishi, 1982, 1983). Freesia corms have been released from dormancy earlier when exposed to smoke produced by burning 260 g of wheat straw on each of four consecutive days (Imanishi and Fortanier, 1983). It has also been recommended that Iris bulbs be exposed to ethylene (which is one of the components of, and produces similar results to, smoke) for 24 hr (Le Nard, 1983; Cascante etal., 1989). Therefore, it is likely that a longer exposure time or a period of exposure times over a number of consecutive days, would have increased the percentage of flowering plants.

The time of year that the smoke treatment was performed may also have been a factor. The season of burning has been found to have an effect on the percentage of plants that flower following a fire. For example, in a study of Xanthorrhoea preissii only 1% of plants flowered following a winter burn compared to 75% following a summer burn (Lamont et al., 2000). A similar result occurred in Xanthorrhoea fulva, with significantly fewer inflorescences produced after a winter burn than a summer burn (Taylor etal., 1998). Wet prairie grass species in South Florida, USA, have also been found to have their flowering promoted by fires during the growing season but not in the dormant season (Main and Barry, 2002). This phenomenon has also been noted in resprouting species such as Stirlingia latifolia (Proteaceae), with post-fire flowering recorded in 92% of plants burnt in summer/autumn compared with 73% of plants burnt in spring (Bowen and Pate, 2004). In the current experiment the treatments were applied during winter, which

424 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica may explain why the flowering percentages were quite low. Had they been performed during summer these percentages may have been increased.

Clipping (or foliage removal) has been found to elicit similar flowering responses as burning in a number of species including Xanthorrhoea australis (Gill and Ingwersen, 1976; Gill, 1981a), Xanthorrhoea fulva (Taylor etal., 1998) and in tallgrass prairie in the USA (Hulbert, 1988). In the current experiment the foliage removal treatment also increased the percentage of flowering D. tasmanica plants compared to the control; and to a similar percentage as the smoke treatment (10% for foliage removal, cf. 12.5% for smoke). Again, although this result was not significantly different to the control treatment, it is still a positive result as it was a 4- fold increase over the control plants. Also, as is the case with burning, the season of foliage removal can be important in determining the extent of floral induction (Brewer and Platt, 1994). For example, only 20-30% of X australis plants flowered after clipping in autumn or winter, compared to 80-97% after clipping in spring or summer (Gill, 1981a). Clipping in summer also resulted in significantly more inflorescences than in winter for X fulva (Taylor et al., 1998). Therefore, as previously mentioned, it is possible that a higher proportion of plants may have flowered had these treatments been applied to plants during summer, rather than in winter.

Burning D. tasmanica plants did not have as much of an effect on flowering as the smoke and foliage removal treatments, although it did produce twice the amount of inflorescences as the control and burnt soil treatments. This was an interesting result as burning is usually more effective than foliage removal for most other species that have been studied. For example, X fulva (Taylor et al., 1998) and the South Florida prairie grass Muhlenbergia capillaris var.filipes (Snyder, 2003). However, it should be noted that a biphasic flowering response has been reported for X australis following burning in May or August (Gill, 1981a), with only approximately 7-25% of plants flowering in the first year, increasing to 70-85% in the second year. It is possible that a similar response may have occurred for D. tasmanica, although perhaps unlikely, as other native monocots such as D. excelsa (Denham and Auld, 1999, 2002) and the more closely related Blandfordia nobilis (Johnson etal., 1994)

425 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica only have a single post-fire flowering event. However, it has been reported that B. nobilis plants burnt in winter or spring will probably not flower until the second summer (Johnson et al., 1994).

The fact that the burnt soil treatment did not elicit the same responses as the smoke, foliage removal and burn treatments suggests that the flowering stimulus for D. tasmanica is not due to changing soil conditions, such as the change in pH from acidic to alkaline that was detected following fire in this experiment. Although most soil characteristics have been reported to show rapid changes in the immediate post- fire period (Specht etal., 1958; Siddiqi etal., 1976; Raison, 1979, 1980; Humphreys and Craig, 1981; Theodorou and Bowen, 1982; Grove etal., 1986; Tomkins etal., 1991; Johnson etal., 1994), and Johnson etal. (1994) reported that the post-fire flowering pulse of B. nobilis appeared to be closely associated with changes in the soil chemistry, this does not appear to be the case for D. tasmanica.

The fact that the very small percentage of plants that flowered in the burnt soil treatment did so quite soon after the treatment was applied (29 DAA) and then there was no further change also suggests that if this treatment did have any effect on flowering it was a relatively short-lived one, possibly due to any stimulatory chemical or nutrient being leached away. It has been reported that the stimulatory chemical in smoke that enhances seed germination in many species is water soluble and thus can easily be leached away (Stewart, 1999b). Interestingly, the soil pH was only changed in the very top layer of the soil (including the ash layer). Lower down in the soil, closer to the root zone, the pH was still very similar to the pre-fire levels. This may also explain why this treatment did not have much of an effect. Also, as the pH following fire was measured on a very rainy day when the soil was quite wet it was still quite alkaline, and perhaps due to some dilution effects from the rain, it may have been even more alkaline just after the treatments were applied.

The first plants to reach anthesis were from the smoke and control treatments. Interestingly, the smoke treated plant had a relatively normal length flowering scape for a flowering D. tasmanica plant, while the control plant only had a very short stem. Therefore, perhaps the control plant had flowered in response to the stress of transplanting and did not have the necessary reserves to produce a flowering stem of

426 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica normal length. It should also be noted that due to the majority of plants having their flowering stem tips eaten by wildlife, it was not possible to ascertain the first treatment in which plants reached anthesis. It is possible that plants other than those mentioned above could have reached anthesis quicker had their stems not been removed.

It should be noted that one deformed flowering stem was noted on a smoke treated plant. Similar deformities, in which stems were unusually short and/or branched into two at the apex and flower buds were larger than normal were also noted in two of the smoke treated plants from the preliminary smoke experiment (Sward, 1995). Although this deformity was only noted in the smoke treated plants in both of these experiments it does not appear to be caused by the smoke treatment. The proportion of plants was very low in the current experiment, and similar deformities have also been noticed in natural populations of D. tasmanica (Curtis, 1952; Sward, personal observation).

In regard to regrowth from the two treatments in which plants were defoliated (foliage removal and burn), regrowth was initially noted in a burn treated plant (71 DAA). However, the majority of regrowth did not occur until 168 DAA, at which time there was regrowth from the same amount of plants in both these treatments. Therefore, regardless of the defoliation treatment applied, plants appear to regrow in a similar time. A similar result occurred when Muhlenbergia capillaris var./Miles from South Florida, USA, was clipped or burnt. Regrowth was similar following both treatments, but burned plants appeared to resprout more vigorously, perhaps due to ash from the fire fertilising the plant (Snyder, 2003).

In conclusion, the results from this experiment suggest that smoke and the removal of foliage are possible stimuli for flowering in D. tasmanica. The importance of smoke as opposed to burning has also been reported for Xanthorrhoea australis. For example, ethylene (a component of smoke) was found to stimulate flowering in this species (Gill and Ingwersen, 1976; Gill, 1981a) and in a later study smoke drift from fires appeared to stimulate flowering up to 200 m from the burnt area (Curtis, 1998). Therefore, it may be possible to force D. tasmanica plants to flower earlier and more profusely using a smoke or foliage removal treatment or a combination of the two.

427 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

However, more research is required to determine whether these treatments can produce significantly more flowering than control or other treatments.

5.5 Summary and Future Research

5.5.1 The effect of cold storage followed by a heat shock on the flowering of Dianella tasmanica

• D. tasmanica plants were kept under cold storage conditions (10°C temp, 8 hr p.p) for 7 months.

• 1/4 of the plants were then transferred to a warm temperature with the same photoperiod (25°C, 8 hr); 1/4 to the warm temperature with a long photoperiod (25°C, 16 hr); 1/4 to the cold temp with a long photoperiod (10°C, 16 hr); and the final 1/4 remained under the original conditions (10°C, 8 hr).

• During the initial cold storage period 16.7% of plants flowered.

• By mid-September 15% of these plants had produced flowering stems.

• Plants growing outside under natural conditions also had flowering stems at this time.

• It took 3 months for plants in the initial treatment to reach anthesis.

• 8.3% of plants died during the initial cold storage treatment.

• At the conclusion of the experiment, the highest percentage of flowering plants occurred in the 25°C, 8 hr treatment (50% of plants flowered).

• 16.7% of plants flowered in the 25°C, 16 hr treatment.

• Plants growing at 10°C under both 8 and 16 hr photoperiods did not flower at all.

• The percentage of plants that flowered in the 25°C, 8 hr treatment was significantly higher than both the 10°C, 8 hr and 10°C, 16 hr treatments; but not the 25°C, 16 hr treatment.

428 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

• The majority of plants in the 25°C, 8 hr treatment developed flowering stems after only 1 month.

• Floral stems were observed on plants in the 25°C, 16 hr treatment, after 1.5 months.

• For plants from both the 25°C, 8 hr and 25°C, 16 hr treatments the time from beginning of elongation of the floral stem to anthesis was approximately 3 weeks.

• At the conclusion of the experiment 4 plants had died - 2 from the 25°C, 8 hr treatment and 2 from the 25°C, 16 hr treatment.

As D. tasmanica plants still flowered in the initial cold storage treatment, it is possible that this temperature was not low enough to prevent the elongation of the floral stem after initiation had occurred. Therefore, it may be worthwhile to use a colder temperature (eg. 5°C) to determine whether this would prevent flowering. Also, in the current experiment plants were not placed into growth cabinets until the end of August, when it is possible that initiation had already occurred (although observations of the apical meristems of other plants that were not used in the experiment suggested that they were still vegetative). Therefore, it would be wise to repeat this experiment and place the plants in growth cabinets earlier (eg. during May) when it would be more likely that floral initiation had not occurred.

It would also be important to determine the effects on flowering of different day and night temperatures as diurnal temperature fluctuations would be experienced by the plants in their natural environment.

Another useful experiment would be to determine whether it would be possible to keep plants under cooler temperatures (thus preventing them from flowering) long term (eg. 12 months), during which time a number of plants could be removed at various times and placed into warmer conditions causing them to flower. If this was possible the opportunity would be available to produce flowering plants at any time of the year that they were required.

Observations of flowering plants in regard to the number of leaves present on flowering, as opposed to non-flowering, plants and the overall size of the plants

429 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica and/or rhizomes would also be important information to obtain. Such information would show what size plants of this species must be before they are competent to flower.

It would also be important to determine the flowering responses to temperature and photoperiod for the rest of the species that have been studied in this thesis, thus providing possible ways of manipulating their flowering behaviour.

5.5.2 The effect of smoke, burning and foliage removal treatments on flowering in Dianella tasmanica

• D. tasmanica plants were subjected to one of five treatments: control (no treatment), smoke, burn, foliage removal and burnt soil, to determine their flowering responses.

• Plants from the smoke, foliage removal and burnt soil treatments had flowering stems 29 DAA.

• Smoke treated plants had the highest percentage of flowering stems at this time (7.5%).

• Control and burn treated plants did not have flowering stems until 42 days after the other treatments (71 DAA).

• The smoke treatment elicited the highest flowering response (12.5%), with the foliage removal treatment producing a similar result (10%).

• Although the percentage of flowering plants from the smoke and foliage removal treatments were 5 and 4 times the amount produced by control plants (respectively), the differences were not significant.

• The burn treatment only caused 5% of plants to flower, but this was still twice the amount of control plants that flowered.

• The burnt soil treatment had the same percentage of flowering plants as the control (2.5%).

430 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

• The response to all treatments was quite rapid, with no change in the percentage of plants with flowering stems beyond 71 DAA.

• Flowers did not reach anthesis until 99 DAA. The first plants to do so were one smoke-treated plant and one control plant.

• The control plant had a very short flowering stem at the time of anthesis, while the smoke-treated plant had a normal length stem.

• One deformed flowering stem was present on a smoke treated plant.

• The majority of regrowth from defoliated plants (from foliage removal and burn treatments) occurred at 168 DAA.

• The same number of plants had regrown new shoots from both foliage removal and burn treatments at this time.

• The soil pH in the experimental area was initially quite acidic (5.5).

• Two weeks after the burning treatments the top soil layer (including ash) was alkaline (9.0). Lower down in the soil profile (below the ash layer) the pH was similar to the initial measurement (6.0).

In order to increase the percentage of flowering plants following the fire-related and foliage removal treatments it would be worthwhile repeating this experiment, with treatments applied in summer rather than winter. The season of burning and clipping has been found to significantly affect the percentage of flowering plants (see section 5.4.2), with summer disturbances producing higher percentages of flowering plants. The current experiment was performed in winter, mainly because this was prior to the normal flowering season for D. tasmanica, and as plants had not begun to initiate flowering, it was fairly certain that the results would not be confounded by plants that were already going to flower regardless of the treatments applied to them.

It may also be worthwhile to increase the exposure time for the smoke treated plants, as the 30 min exposure time used in the current experiment was quite short compared to recommendations for ornamental bulb and corm species (see section 5.1.5). It is also probably shorter than plants would be exposed to during a bushfire event, when burnt material may continue to smoulder for days or even weeks following the fire.

431 Chapter 5. The effect of manipulation of environmental parameters on flowering in D. tasmanica

Therefore, a longer exposure time or a range of exposure times would be worth trying.

Further investigation of the smoke effect would also be worth pursuing, as the effects of this component of fire appear to have been downplayed in regard to the flowering response. Ethylene (a component of smoke) appears to elicit flowering responses in a range of species, both Australian and ornamental bulb and corm species. Therefore, an experiment that included an ethylene treatment could be worthwhile. Ethephon, a substance that produces ethylene after uptake by a plant (Kamerbeek and De Munk, 1976), has also produced similar results in ornamental bulb species, and can be sprayed onto plants, meaning that it is easier to handle than a gas. Therefore, a spray with Ethephon would perhaps be another suitable treatment. In light of the recent discovery of the chemical in smoke that stimulates germination in many native Australian plant species (Flematti et al., 2004), it would also be worthwhile testing whether this chemical could also stimulate flowering in D. tasmanica.

To determine whether D. tasmanica has a single flowering response following fire, like other native monocot species, or whether its response may extend over a number of seasons, like some other resprouting species, it would also be useful to extend the experiment over a number of years, rather than just the first flowering season following the fire that this experiment was limited to.

Another possible experiment would be to determine the relationship between plant size and flowering, and to determine whether burning and/or smoke could induce plants that were of sub-critical size to flower, as has been reported for other species including Xanthorrhoea preissii (Lamont et al., 2000), and some ornamental flower bulb and corm species including Iris (Schipper, 1982) and Narcissus tazetta (Hanks, 1993).

432 Chapter 6 General Conclusions

The various investigations in this study have shown that Blandfordia, Dianella, Milligania, Diplarrena and Isophysis do indeed possess characteristics that make them suitable for the horticulture industry.

Mainland Australian species of Blandfordia, in particular B. grandiflora, have already proven to be successful as cut flowers in both domestic and overseas markets (Gorst, 1996; Johnson, 1996a) and the information gained from this study suggests that the Tasmanian endemic B. punicea would also be suitable. Propagation through the conventional method of seed germination was very successful, with 90-100% of seeds germinating without the need for any seed pre-treatments. However, as it can take up to three years for plants grown from seed to reach flowering (Greig, 1993; Johnson, 1987, 1996; Wrigley and Fagg, 1996; Page and Olds, 2004), and clonal plants will not be produced, it was necessary to investigate the use of in vitro propagation for this species.

B. punicea was found to be relatively easy to grow in vitro and responded similarly to other Blandfordia species under these conditions. Corm pieces were found to be unsuitable initial explants due to problems with contamination. However, immature floral explants such as flower buds, floral scape sections and pedicel/pedtmcle junctions all proved to be excellent explant sources. They were easy to disinfest and the latter two in particular, were highly regenerative when grown on FBM, a flower bud initiation medium (1/2 MS + 1011M BAP + 0.5gM IBA) used successfully for B. grandiflora (Bunn and Dixon, 1997). Shoots also multiplied well, with rates as high as 36.3 x achieved on FBMM, a shoot multiplication medium also developed for B. grandiflora (1/2 MS + 2.51AM kinetin + 0.251.1M BAP) (Bunn and Dixon, 1997). Roots also developed on shoots growing on this medium, meaning that a separate

433 Chapter 6. General Conclusions

rooting medium was not required. B. punicea rooted plantlets were transplanted successfully to ex vitro conditions, with minimal losses (mainly due to fungal infestation caused by over-watering). The successful use of floral explants means that it would be possible to select mature plants, with known attributes, as stock plants and thereby ensuring that the clones to be produced will have suitable characteristics for horticulture. For example, B. punicea, like the other Blandfordia species, exhibits a wide range of flower colours and it would be possible to select favourable variants for commercial production. As well as its possible use as a cut flower, B. punicea is also suited to landscaping applications as a rockery plant (Moore etal., 1993; Wrigley and Fagg, 1996), grown in drifts (Greig, 1990, 1993) or mass plantings (Greig, 1993). It is also suitable as a pot plant specimen (Blombery, 1972; Moore etal., 1993; Johnson, 1996a; Stewart, 2002).

Although a lot of work has been done to develop the Blandfordia genus as a commercial crop, still much remains to be done, especially in regard to its flowering behaviour. More specifically, the effects of photoperiod and temperature on flowering is important information that could lead to the manipulation of flowering in order to suit specific target markets. Some work has been done on the effects of temperature on flowering (Goodwin and Watt, 1994; Goodwin etal., 1995). However, with more information it may be possible to manipulate flowering so that plants can be forced to flower for specific dates, or indeed, all year-round so that flowering pot plants and/or cut flowers can be produced for a wider range of markets than is currently possible.

Within the last few years the popularity of the Dianella genus has increased, particularly due to the introduction of the "Border" seriesTM of D. ensifolia by Anthony Tesselaar International in 2002. D. tasmanica also has many characteristics that are suitable for horticulture. Not only is it one of the few blue-flowered native plants, but it is also useful as a foliage plant, with its large flax-like leaves (Wrigley and Fagg, 1983, 1996). The long-lasting bright blue berries may sometimes be more ornamental than the flowers (Page and Olds, 2004). Its clump-forming growth habit means that it is particularly useful in rockeries (Elliot and Jones, 1984; Wrigley and

434 Chapter 6. General Conclusions

Fagg, 1983, 1996) and as a container plant (Elliot and Jones, 1984). Although this species has a low natural germination percentage (38%) and has dormancy mechanisms present, it was possible to increase the percentage germination using a number of methods. Smoke treatments, both direct and in liquid extract form, were promising, with the percentage of seeds germinating increasing with increasing smoke extract concentration. However, undiluted (100%) smoke inhibited germination. The best treatment for this species was removal of part of the testa under sterile conditions and growth in vitro, with 90% germination achieved. This result suggests that the main dormancy mechanism preventing D. tasrnanica seeds from germinating is a physical dormancy caused by the hard testa.

As conventional methods of propagating D. tasmanica by division are slow, in vitro propagation was also investigated for this species. Initial experiments had shown that D. tasmanica could be grown in vitro using rhizome pieces as initial explant types (Sward, 1995). Therefore, this explant type was initially used for further experimentation. A complex disinfestation procedure was required to effectively sterilise rhizome pieces, and when successful, this explant type grew well on a basal MS. The best shoot multiplication was achieved when intact explants were grown on MS + 321AM BAP; while subcultured shoots multiplied best on MS + 81./M BAP. The best rooting medium was MS + 2p1M NAA. Almost 100% of shoots in this treatment grew roots, with an average of 14.4 roots per shoot. Rooted plantlets were successfully transplanted to ex vitro conditions, with minimal losses (again, mainly due to damping off caused by over-watering). Due to the contamination problems associated with the underground rhizome explants, immature floral explants were also tried. Flower bud and floral scape section explants were easier to surface sterilise, with only low contamination rates occurring. Flower buds were quite regenerative, but scape sections were somewhat unpredictable. Somatic embryogenesis was also investigated as a more rapid method for in vitro propagation of D. tasmanica. Preliminary results were promising, but more work is required to determine how effective this method could be. In this study, D. revoluta var. revoluta and D. intermedia were also propagated in vitro

435 Chapter 6. General Conclusions for the first time. Using flower buds and floral scape sections as initial explants, preliminary results were promising. Although shoots were not obtained from D. revoluta var. revoluta flower buds, callus was obtained. For D. intermedia results were even more encouraging, with shoots obtained from both flower bud and floral scape section explants, with the latter being the most regenerative.

DianeIla tasmanica has also shown an ability to flower "on demand" when environmental conditions are manipulated. By keeping plants under conditions that were unfavourable for floral initiation and development, they could be maintained in a vegetative state. It was then possible to force them to flower by placing them in a short photoperiod (8 hr) at a constant high temperature (25°C). Flower forcing is an important factor in the horticulture industry as there is often a demand for flowering plants all year-round. This potential cannot be realised for many bulbous species as they possess a dormancy period as part of their life cycle. However, this problem does not occur in the rhizomatous DianeIlas which do not have a dormancy period, and with their ability to flower "on demand" the possibility of producing flowering plants all year-round may be realised in the future. The fire-related treatments of smoke and foliage removal also increased the percentage of flowering plants compared to the control. However, the time taken to reach anthesis was much longer than in the short day, high temperature treatment. Therefore, the growth cabinet treatment, or perhaps a combination of this and the smoke treatment, should be the chosen method for a commercial operation where time and efficiency are important factors.

Milligania densiflora is a very attractive and highly desirable species for cultivation, but currently it is not often grown in Australian gardens (Elliot and Jones, 1993). It naturally occurs at high altitudes (above 700 m) and is often subjected to extremely low temperatures (Elliot and Jones, 1993), resulting in very frost-hardy plants (Wrigley and Fagg, 1996). This characteristic means that the species has excellent potential for cool temperate regions (Elliot and Jones, 1993). The creamy white flowers are attractive, but the plants are also recommended for their foliage (Wrigley

436 Chapter 6. General Conclusions and Fagg, 1996). As a garden plant this species is best suited to rockeries (Wrigley and Fagg, 1996) and it also grows well as a container plant (Elliot and Jones, 1993).

Similar to D. tasmanica, M densiflora also had quite a low natural germination percentage (48%). However, this percentage could be increased to 88% simply by germinating the seed in vitro. The best germination (90%) was achieved by disinfesting seeds in 2% Na0C1 for 35 min and growing them on 1/2 MS. In vitro propagation was also investigated for this species, which had never been propagated in this manner before. Root pieces and shoot and leaf bases were unsuccessful initial explant sources, but whole immature flower buds were promising. Although shoots were not obtained from the buds, some regeneration (which appeared to be the start of shoot development) did occur. This explant type should be investigated further as it is likely that the incorrect developmental stage was used.

Seedlings germinated in vitro were used for further micropropagation studies due to the lack of regeneration from the other explant types tried. Although seedlings are not an ideal source of explant material for a micropropagation system where clones are the expected outcome, it was hoped that the results from this experiment could be employed for micropropagation of shoots from other explant types in the future. The best shoot multiplication occurred on 1/2 MS + 8uM BAP. One hundred percent of seedlings growing on this media produced shoots, the mean number of shoots per seedling was the highest and shoot survival was also very good. Roots could be induced on shoots when grown on 1/2 MS + 1 gl.,"1 AC; and rooted plantlets adapted well to ex vitro conditions. Almost 90% were still alive 1 month after transplanting.

The Diplarrena genus (containing D. moraea and D. latifolia) is showy and excellent for cultivation (Elliot and Jones, 1984). Similar to the South African Dietes, their clumping nature means that they are ideal when mass planted as a border (Greig, 1993), in rock gardens or beside a water feature (Greig, 1993; Wrigley and Fagg, 1996; Barnard et al., 2004). They are also excellent container plants (Blombery, 1972; Elliot and Jones, 1984; Greig, 1993). Selections of D. latifolia such as "Amethyst Fairy" are relatively popular in Australia and are also available in

437 Chapter 6. General Conclusions

Europe (Elliot and Jones, 1984). Within Tasmania, D. moraea is often used by local councils as a landscaping plant.

D. moraea had a very high natural germination percentage (97%) and therefore can easily be germinated without the need for any seed pre-treatment. However, in order to rapidly multiply selected clones it would be necessary to propagate them in vitro. Seeds were used as an initial explant source, with seedlings used for multiplication media experiments. D. moraea seedlings produced very high numbers of new shoots on MS + 1281AM BAP and MS + 12811M 2iP. However, the majority of these shoots also developed callus. All shoots growing on treatments containing low concentrations of cytokinin and the control also grew roots. Rooted plantlets were successfully transplanted to ex vitro conditions, with no losses in the first month post-transplanting. Although these experiments with D. moraea were only preliminary, the results have provided clear direction for future research using other more suitable explant sources.

D. latifolia had a reasonably high natural germination percentage (76%), which could be increased to 89% by germinating seed in vitro on MS + 1281AM ldnetin. D. latifolia was also successfully propagated in vitro using seeds and flower buds as initial explant sources. Seedlings were used for multiplication media experiments, with the greatest proliferation of new shoots occurring when they were grown on MS + 3211M BAP and MS + 321.1M 2iP. Roots grew on the majority of shoots growing on low concentrations of cytokinin as well as the control treatment. Rooted plantlets were successfully transplanted to ex vitro conditions (97% of plantlets were still alive one month after transplanting). Preliminary experimentation using immature flower buds showed that shoot regeneration was possible. However, more work is required to ascertain the correct stage of development to subsequently produce the greatest number of shoots.

Isophysis tasmanica is an unusual, but stunning species. Due to the large size and unusual colour of the flower it is highly desirable for horticultural purposes. Also, the plant itself is quite small, making it an ideal candidate for a small potted plant. Due to high freight costs associated with exporting potted plants to overseas markets,

438 Chapter 6. General Conclusions there is a general demand for plants to be under 30 cm in height (Worrall, 1996a). Most of the existing pot plant varieties of Anigozanthos, for example, require the use of growth regulators to achieve this size, generally resulting in much longer production times (Worrall, 1996a). However, this would not be necessary for tasmanica as it is naturally a small plant. This species would also be an excellent rockery plant (Wrigley and Fagg, 1996) or associated with a water feature.

I. tasmanica was very easy to propagate from seed, with 90-100% of seeds germinating without any pre-treatment. I. tasmanica was also propagated in vitro for the first time, using seeds as initial explants. Whole immature flower buds were also tried, but did not respond well to in vitro conditions, most likely because they were too mature at the time they were cultured. Using seedlings for multiplication media experiments, shoots multiplied best on MS + 32p.M BAP. Shoots were induced to form roots on MS + 1 gE 1 AC. Transplanting to ex vitro conditions was initially quite successful, but this species appears particularly susceptible to rotting if the soil becomes too wet, so they must be very carefully monitored in future micropropagation experiments.

This investigation into the horticultural potential of native Tasmanian Liliaceae and Iridaceae has shown that these species do indeed possess characteristics that make them suitable for use as ornamental plants and/or cut flowers. All could be propagated by seed, and for those with low natural germination percentages (Dianella tasmanica and Milligania densiflora) it was possible to identify the reasons behind these low natural levels, and therefore determine methods which could increase these to acceptable levels for commercial situations. Propagation from seed can be a slow process, with many species taking years to reach flowering size. However, this method still plays an important role in breeding programs to develop new varieties, and therefore, should not be overlooked.

To produce plants, such as new varieties, in commercial quantities a fast and reliable method of propagation is necessary. Therefore, this investigation included micropropagation as a possible means of mass-producing these plants, the majority of which had never previously been grown in vitro. Despite some of the species (eg.

439 Chapter 6. General Conclusions

Isophysis tasmanica) being labelled as difficult to propagate conventionally, all adapted very well to in vitro conditions, and multiplied rapidly. Successful protocols were devised for disinfestation, shoot initiation and multiplication, rooting and transplanting to ex vitro conditions. However, it should be noted that more research is still required to refine some of these protocols. In general, results were promising and showed that it was definitely possible to propagate all species using this method.

Before these species could be introduced to commercial horticulture, more research into their growth requirements once transplanted from tissue culture would definitely be required. Information of this kind is available for Blandfordia (see Lamont etal., 1990) due to the large amount of research that has already gone into the commercialisation of this genus. Limited information is also available for Dianella and Diplarrena, which have also been commercialised to some degree. However, for genera such as Milligania and Isophysis, perhaps due to their endemicity to Tasmania and restricted distributions within the state, very little information is currently available. Therefore, more research would be necessary to ensure that the growth requirements of these plants are more fully understood. This study has shown that Isophysis in particular is very susceptible to rotting when over-watered, and therefore would require extremely well-drained soil.

Information gained in regard to the influence of temperature and photoperiod on flowering in Dianella tasmanica was also very important, and similar experiments should be carried out on the other species to determine factors involved in floral initiation and development. Such information would mean that these species may also be forced to flower out of season or even all year-round.

With the increase in popularity of Australian native plants, such as the monocots included in this study, also comes the need for protection and conservation of their natural populations and habitats. Practices such as bush-picking should be discouraged, while further research into their propagation and cultivation should be encouraged. This will ensure that not only are the natural populations protected, but also that material of the highest quality is available for commercial markets.

440 Chapter 6. General Conclusions

Techniques such as in vitro propagation are essential in such cases as rapid multiplication can be achieved, meaning that new varieties can reach the market place in much shorter times than were previously possible using conventional methods. This is especially important for recalcitrant species and those that reproduce slowly under natural conditions, such as the native monocots in this study. In vitro propagation is also an excellent method in terms of conservation, as only very small plant parts are required to initiate cultures, meaning that there is minimal disturbance to both the plant itself and the surrounding vegetation.

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494 Appendices

Appendix 2.1. Smoke extract solution methodology

A concentrated smoke extract solution, or eluate, was made by bubbling smoke produced from burning native vegetation through a 2 L container of distilled water for approximately 1 hr. The combustion of the vegetation took place in a metal drum to which an inlet was fitted. Air was pumped in through the inlet, and smoke was pumped out through an outlet connected to a hose that was placed in the container of distilled water. The resultant concentrated solution was stored in a cold room at 4°C until required. Solutions of 1%, 10% and 50% smoke extract were made by diluting the concentrated stock with distilled water.

495 Appendices

Appendix 2.2. Constituents of Murashige and Skoog (MS) Medium (1962).

Component Chemical compound Concentration Macronutrients NH4NO3 20 mM

KNO3 18.8 mM

CaCl2.2H20 4 mM MgSO4.71-120 1.5 mM

KH2PO4 1.2 mM Micronutrients MnSO4.4H20 100 [tM

ZnSO4.7H20 3011M 113B03 1001AM KI 5 1.IM Na2Mo04.2H20 11.1M

CuSO4.5H20 0.11AM

CoC12.6H20 0.11.1M

Iron Na2EDTA 100 [tM

FeSO4.7H20 1001.1M

Growth Factors Nicotinic acid 4 uM Pyridoxine.HC1 2.4 1..tM Thiamine.HCI 0.3 uM Glycine 26.6 laM Inositol 555 p.M Carbon Source Sucrose 30 g/L (8.7 mM) pH 5.5-5.8

496 Appendices

Appendix 2.3. Constituents of Embryo Culture (EC) Medium

Component Chemical compound Concentration Macronutrients NH4NO3 20 niM

KNO3 18.8 mM

CaC12.21-120 4 mM

MgSO4.71-120 1.5 mM

ICH2PO4 1.2 mM Micronutrients MnSO4.4H20 1001AM

ZnSO4.7H20 301AM

H3B03 100 p,M

K1 5 ;AM Na2Mo04.2H20 1 j.tM

CuSO4.5H20 0.1 i_tM

CoC12.6H20 0.1 p,M

Iron Na2EDTA 100 p,M

FeSO4.7H20 100 p,M

Growth Factors Nicotinic acid 41.IM Pyridoxine.HCI 2.4 1..tM Thiamine.HC1 0.3 1.tM Glycine 26.611M Inositol 555 1.1M

PGR NAA 0.3 pM Carbon Source Sucrose 40 g/L (11.6 mM) pH 5.5

497 Appendices

Appendix 3.1. Native Potting Mix (Department of Plant Science Glasshouse)

For 45 litres of Basic Mix (1 part = 1 shovel full):

6 parts composted Pine bark 4 parts coarse washed river sand 1 part peat moss 45 g 3-4 month Osmocote® N: P: K-19: 2.6: 10 90 g 8-9 month Osmocote® N: P: K-17: 1.6: 8.7 121 g dolomite lime 23 g Micromax® 13.5 g iron chelate

498 Appendices

Appendix 3.2. NN (Narcissus Initiation) media constituents

Component Chemical compound Concentration Macronutrients NH4NO3 20 mM KNO3 18.8 mM CaC12.2H20 4 mM

MgSO4.7H20 1.5 mM

KH2PO4 1.2 mM Micronutrients MnSO4.4H20 100 [tM ZnSO4.7H20 301.1.M

H3B03 100 i.tM KI 5 }.tM Na2Mo04.2H20 1 uM CuSO4.5H20 0.1gM CoC12.6H20 0.111M

Iron Na2EDTA 1001AM FeSO4.7H20 100 ;AM

Growth Factors Nicotinic acid 1.6 i_tM Pyridoxine.HCI 0.96 IIM Thiamine.HC1 0.12 ?AM Glycine 10.64 pM Inositol 555 1.1M PGRs NAA 5.4 uM BAP 44.4 pA4 Other ingredients Casein hydrolysate 0.4 g/L Carbon Source Sucrose 30 g/L (8.7 mM) pH 6.0

499 Appendices

Appendix 3.3. NM (Narcissus Multiplication) media constituents

Component Chemical compound Concentration Macronutrients NI-14NO3 20 mM KNO3 18.8 InM CaCl2.2H20 4 mM

MgSO4.7H20 1.5 mM

KH2PO4 1.2 mM Micronutrients MnSO4.4H20 100 1.tM

ZnSO4.7H20 30 p.M H3B03 1001.1M KI 5 i.IM Na2Mo04.2H20 1 AM CuSO4.5H20 0.1 uM CoC12.6H20 0.1 p,M

Iron Na2EDTA 1001.IM

FeSO4.7H20 100 uM

Growth Factors Nicotinic acid 4 uM Pyridoxine.HCI 2.4 uM Thiamine.HC1 0.3 uM Glycine 26.6 I.LM Inositol 555 i.tM PGRs NAA 10 uM BAP 9 ;AM Carbon Source Sucrose 30 g/L (8.7 mM) pH 5.5

500 Appendices

Appendix 3.4. Observations of B. punicea whole flower bud explants at 4 different developmental stages, 1.5 months after placement on MS and FBM.

Developmental MS FBM stage 10 buds - no regeneration. 27 buds - no regeneration. All had expanded in size and 26 had expanded in size and were were green, 5 of these were still green, 5 of these were starting to starting to turn yellow, turn yellow. 1 bud was brown - appeared dead. 2 5 buds - all had turned brown 15 buds - 3 had shoots regenerating and appeared to be dying. from the base of the bud (I had only 1 had developed a seed capsule just begun to grow; 1 had 1 large which had started to turn shoot and 2-3 small ones starting to brown. grow; the other had a small shoot. 3 had swollen at the ovary The remaining 12 buds were still region (which was still green). green and had expanded in size. 10 buds - no regeneration. 48 buds - 2 had regenerated shoots All had expanded but had from the base of the bud. turned brown and appeared to 45 had expanded in size and were be dying. still green (but no regeneration). 1 bud was turning brown and was contaminated. 4 5 buds - all had turned brown 14 buds - no regeneration. and looked dead, but pedicels 10 had turned brown and appeared were still green. to be dying. 1 bud was contaminated. 4 were still green.

501 Appendices

Appendix 3.5. Observations of B. punicea floral explants 1 month after disinfestation and initiation of cultures.

Explant type Developmental Observations stage Whole flower buds 1 5 explants -2 had bulbous structures just starting to form at the base of the pedicel. 2 7 explants -2 had bulbous structures just starting to form at the base of the pedicel. 3 3 explants - 0 had shoots. No change in explant appearance. 4 9 explants - 0 had shoots. Buds had swollen. 5 5 explants - 0 had shoots. Buds had swollen. Floral scape sections N/A 25 explants - 15 had started to produce bulbous structures; 7 hadn't changed in appearance; 3 were dead. Pedicel/peduncles N/A 4 explants - all had started to produce bulbous structures.

Appendix 3.6. Observations of B. punicea floral explants approximately 1.5 months after disinfestation and initiation of cultures.

Explant type Developmental Observations stage Whole flower buds 1 5 explants - 3 had turned brown, but pedicels were still green (2 of these had bulbous structures at the base of the pedicel); 1 had formed a green capsule; 1 was starting to turn brown (no regeneration). 2 7 explants - 5 had shoots forming at the base of the pedicel; 2 had no change in appearance. 3 3 explants - 1 appeared to be starting to grow shoots; 2 had no change in appearance. 4 9 explants - 0 had shoots. Buds had swollen and some had split open, but no regeneration. 5 5 explants - 0 had shoots. Buds had swollen and some had split open, but no regeneration. Floral scape sections N/A 22 explants - 20 had produced shoots (2 of these were contaminated); 2 without shoots were also contaminated; 18 were still ok and some of the shoots had grown quite large. Pedicel/peduncles N/A 4 explants - all had produced shoots that were now quite large.

502 Appendices

Appendix 3.7. Observations of B. punicea floral explants approximately 3.5 months after disinfestation and initiation of cultures.

Explant type Developmental Observations stage Whole flower buds 1 5 explants - 4 had started to produce shoots; 1 had a green capsule that was starting to turn brown at the tip (no shoot regeneration). 2 7 explants - 6 had shoots forming at the base of the pedicel; 1 had no shoots. 3 3 explants - 2 had started to develop shoots. 4 9 explants - 5 had started to develop shoots. 5 5 explants - 2 had started to develop shoots. Floral scape sections N/A 18 explants - all had produced shoots and some of the shoots had grown quite large. Pedicel/peduncles N/A 4 explants - all had produced shoots that were now quite large.

Appendix 3.8. Observations of B. punicea whole flower bud explants approximately 5 months after disinfestation and initiation of cultures. Pedicel/peduncle and floral scape section explants had already been transferred to multiplication media.

Explant type Developmental Observations stage Whole flower buds 1 5 explants - all had shoots; 3 also had callus forming. 2 7 explants - all had shoots; 5 also had callus forming; 1 was contaminated. 3 3 explants - 2 had shoots and callus. 4 9 explants - 5 had shoots and callus; 3 had callus only. 5 5 explants - 3 had shoots and a small amount of callus.

503 Appendices

Appendix 3.9. Observations of D. tasmanica flower bud and flower stem section explants grown on MS and FBM for 1 month. FB = Flower Bud; FSS = Floral Scape Section.

Explant ID MS FBM and type AR1 - FB 4 explants - no change. 4 explants - all were expanding in size and were very green. AR2 - FB 5 explants - no growth and were 5 explants - buds were expanding in starting to turn brown. size and were very green. AR2 - FSS 4 explants - still green but no 4 explants - still green but no growth. growth. AR3 - FB 6 explants - still green but no 6 explants - buds were expanding in growth; size and were very green; 1 contaminated. 1 contaminated. LR1 - FB 5 explants - all still green; 2 5 explants - all still green; 4 were buds had expanded slightly, expanding in size. LR2 - FB 9 explants - 8 were still green 11 explants - 10 were still green and and were expanding in size; 1 buds were expanding; 1 had turned had turned brown. brown. LR2 - FSS 12 explants - all still green but 12 explants - all still green but no no growth. growth. LB1 - FB 12 explants - 5 were still very 12 explants - 11 were still green and green but no change; 7 were buds were expanding in size; starting to turn brown. 1 was contaminated and dead. LB1 - FSS 12 explants - all still green but 13 explants - all still green but no no growth. growth.

504 Appendices

Appendix 3.10. Observations of D. tasmanica flower bud and floral scape section explants grown on MS and FBM for 3 months. FB = Flower Bud; FSS = Floral Scape Section.

Explant ID MS FBM and type AR1 - FB 4 explants - 1 had a green 4 explants - 3 had shoots. swelling (perhaps the start of shoot development); 3 were dead. AR2 - FB 5 explants - all were dead. 5 explants - all had shoots. AR2 - FSS 4 explants - all were still green; 1 4 explants - all were still green, but was starting to turn black. no shoot development. AR3 - FB 5 explants - 4 had green pedicels, 5 explants - all had shoots. but buds were brown; 1 was dead. LR1 - FB 5 explants - 1 looked like it may 5 explants - 2 had shoots; 1 (with no be developing a shoot; 4 had shoots) was dead - was a very small turned brown. terminal bud. LR2 - FB 9 explants - 1 had a small amount 11 explants - 10 were producing of green and was (perhaps) shoots; I was dead. developing a shoot; 8 were dead. LR2 - FSS 12 explants - all had turned brown 12 explants - 3 were still green; 9 and were dead. were dead. LB1 - FB 12 explants - 5 were still green (1 11 explants -9 were still green (8 had produced a shoot); 7 were were producing shoots); 2 were brown and dead. brown and dead. LB1 - FSS 12 explants - all had turned brown 13 explants - all had turned brown and were dead. and were dead.

505 Appendices

Appendix 3.11. Observations of D. tasmanica flower bud and floral scape section explants grown on MS and FBM for 5 months. FB = Flower Bud; FSS = Floral Scape Section.

Explant ID MS FBM and type AR1 - FB 1 explant - the remaining explant 4 explants - all had shoots; 2 explants was still slightly green, but no both had 2 long shoots. shoots had developed. AR2 - FB 5 explants - all were dead. 5 explants - all had shoots; 3 had shoots that were quite large. AR2 - FSS 4 explants - 3 were still green, but 4 explants - all were brown and dead. had no shoots; 1 was dead. AR3 - FB 4 explants - all were dead. 5 explants - all had shoots; 4 were still quite small; 1 had very long shoots (-, 4 cm long). LRI - FB 5 explants - 1 was developing 2 4 explants - 2 had shoots; the other 2 small shoots, but bud was mainly were still green but no regeneration. brown; 4 were dead. LR2 - FB 1 explant - the 1 remaining 10 explants - all had produced explant had a shoot, and also 1 shoots; 4 were now dead. root. LR2 - FSS 12 explants - all were dead. 3 explants - I was still green; 2 were dead. LB1 - FB 5 explants - 1 had 2 long green 9 explants - all were still green, 8 had shoots; 4 were dead. small shoots; 1 was still green but no regeneration. LB I - FSS N/A N/A

506 Appendices

Appendix 3.12. Observations of D. tasmanica flower bud and floral scape section explants (7 months after cultures were initiated) that remained on MS and FBM after those with large shoots were transferred to multiplication media. FB = Flower Bud; FSS = Floral Scape Section.

Explant ID MS FBM and type AR1 - FB 1 explant - the remaining explant 2 explants - both had 2 small had turned brown and was dead. shoots. AR2 - FB N/A 1 explant - had small shoots developing. AR2 - FSS 3 explants - 1 was still slightly 4 explants - all were brown and green; 2 were dead. dead. AR3 - FB N/A N/A LRI - FB 1 explant - had developed 2 small 2 explants - both were still green shoots, but bud was mainly brown. but no shoot regeneration (I was covered with callus). LR2 - FB N/A 2 explants - both were dead (1 had shoots). LR2 - FSS N/A 1 explant - the remaining explant was dead. LB 1 - FB N/A 6 explants - 2 were still green and had produced shoots (the shoots on 1 were dead but rest of bud still alive); 4 were dead. LB 1 - FSS N/A N/A

507 Appendices

Appendix 3.13. Observations of D. tasmanica flower bud explants after growing on FBM for 1 month.

Explant II) Flower buds Floral scape 1 1(1) 6 explants - all still green. 2 had produced 1 shoot; 3 had produced immature fruit (1 was starting to turn blue). 1(2) 1 explant - still green and had started to produce I shoot. 1(3) 3 explants - all still green. 1 had expanded slightly but no regeneration; 2 had started to produce immature fruit. 1(4) 4 explants - 1 had produced 1 shoot and an immature fruit; 2 had produced immature fruit (1 was turning blue); I had no regeneration and had turned yellow. 1(5) 4 explants - all still green and had produced immature fruit (2 fruit were still green and 2 had a blue tinge). 1(6) 6 explants - all still green. 1 had started to produce 2 shoots; 5 had produced immature fruit (4 fruit still green, I had a blue tinge). 1(7) 7 explants - 5 had produced immature fruit (4 fruit still green, 1 had turned blue/purple); I was dead; 1 was contaminated. 1(8) 5 explants - 3 had immature green fruit; 1 was dead (had dropped off pedicel); 1 was contaminated. 1(9) 10 explants - 3 had produced 1 shoot (1 of these also had developed an immature bud); 5 had immature green fruit; 1 was still green but no regeneration; 1 was dead (dropped off pedicel). 1(10) 7 explants - 1 had produced 1 shoot; 4 had produced immature green fruit; 1 was still green but no regeneration; 1 was still partially green but starting to turn brown. Floral scape 2 2(1) 6 explants - 5 had produced immature green fruit; 1 was still green but no regeneration. 2(2) 3 explants - 1 had started to produce immature green fruit; 1 was dead (dropped off pedicel); 1 was contaminated. 2(3) 4 explants - 3 had produced immature green fruit; 1 was dead (dropped off pedicel). 2(4) 5 explants - I had produced a shoot and an immature green fruit; 4 had produced immature green fruit. 2(5) 6 explants - 1 had produced a shoot; 4 had produced immature green fruit; 1 was still green but no regeneration. 2(6) 8 explants - 7 had produced immature fruit (6 still green, 1 with a blue tinge); 1 bud had turned yellow/brown and had opened. 2(7) 9 explants - 1 had produced a shoot; 6 had immature green fruit; 1 was dead; I was contaminated. 2(8) 10 explants - 1 had produced 2 shoots and an immature green fruit; 7 had immature green fruit; 1 was dead; l had dropped off pedicel, was still green but no regeneration. 2(9) 22 explants - 10 had produced shoots (1 also had an immature fruit); 6 had produced immature fruit (5 green, I had turned blue); 3 were still green but no regeneration; 2 were dead (1 dropped off pedicel); 1 was contaminated.

508 Appendices

Appendix 3.14. Observations of D. tasmanica flower bud explants after growing on FBM for 3 months.

Explant ID Flower buds Floral scape 1 1(1) 6 explants - 2 had produced 1 shoot (1 was dead; the other also had a developing fruit - had broken off pedicel and was shrivelling); 1 still had a green pedicel, but bud was brown; 3 had produced blue fruit. 1(2) 1 explant - had produced 1 dark green shoot. 1(3) 3 explants - 2 had produced green shoots (1 also had 2 blue fruit); 1 had produced a large blue fruit. 1(4) 4 explants - 1 had produced 1 shoot; 3 were dead. 1(5) 4 explants - 1 had produced a shoot and a blue fruit; 3 had produced blue fruit. 1(6) 6 explants - 1 had produced 2 shoots; 3 had produced blue fruit (1 had 2 fruit); 2 had produced immature fruit that had died. 1(7) 5 explants - 1 had produced a shoot and a blue fruit; 2 had produced blue fruit; 2 had immature green fruit (1 has 2 fruit). 1(8) 3 explants - all had produced immature fruit with a blue tinge. 1(9) 9 explants - 3 had produced shoots and fruit (2 blue and 1 still green) ; 3 had produced blue fruit; 1 had an immature green fruit; 2 were dead. 1(10) 7 explants - 1 had produced 1 shoot and a blue fruit; 3 had produced immature green fruit (1 had 2 fruit); 3 were dead (1 had a shoot). Floral scape 2 2(1) 6 explants - 2 had produced blue fruit; 2 had produced immature fruit that had died; 2 were dead. 2(2) 1 explant - had produced a blue fruit. 2(3) 3 explants - 1 had produced a blue fruit; 2 had produced immature green fruit that had died. 2(4) 5 explants - 1 had produced a shoot and a blue fruit; 4 had produced blue fruit. 2(5) 6 explants - 1 had produced a shoot + an immature green fruit; 3 had produced blue fruit (1 was dead); 1 had produced an immature green fruit; 1 was dead. 2(6) 8 explants - 7 had produced blue fruit; 1 was dead. 2(7) 7 explants - 1 had produced a shoot; 4 had produced blue fruit (1 was dead); 1 had produced 2 immature green fruit; 1 was dead. 2(8) 9 explants - 2 had produced shoots (1 was partly dead); 5 had produced blue fruit; 2 were dead. 2(9) 19 explants - 12 had produced shoots (3 were now dead; and 4 also had produced blue fruit - 3 had 2 fruit each); 4 had produced blue fruit; 3 were dead.

509 Appendices

Appendix 3.15. Observations of D. tasmanica leaf segment explants grown on 3 different callus-inducing media in the light and the dark for 1 month.

Treatment Rep Light Dark 1. MS+5111■4 2,4-D 1 20 explants - 3 had possible 20 explants - 2 had possible callus; 2 were still green. callus; all were brown. 2 20 explants - 2 had possible 20 explants - 4 had possible callus; 1 still green. callus; 5 still green. 3 20 explants - 2 had possible 20 explants - 1 had possible callus; 3 still green. callus; 3 still green. 2. MS+101AM 2,4-D 1 20 explants - none appeared to 20 explants - none appeared to have any callus; 2 still green. have any callus; all were brown. 2 20 explants - 1 had possible 20 explants -3 had possible callus; all were brown. callus; 3 were partially green. 3 20 explants - none appeared to 20 explants -4 had possible have any callus; all were callus; all were brown. brown. 3. MS+20),IM 2,4-D 1 20 explants - 1 had possible 20 explants - 1 had possible callus; all were brown. callus; all were brown. 2 20 explants - none appeared to 20 explants - none appeared to have any callus; 2 still green. have any callus; 3 were partially green. 3 20 explants - none appeared to 20 explants -2 had possible have any callus; 2 were callus; all were brown. partially green.

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Appendix 3.16. Observations of D. tasmanica leaf segment explants grown on 3 different callus-inducing media in the light and the dark for nearly 2 months.

Treatment Rep Light Dark 1. MS+51AM 2,4-D 1 20 explants - 3 had callus 20 explants - none had callus; all and 2 of these were were brown. developing roots with many root hairs. 2 20 explants - I had callus; 20 explants - 3 had callus and 1 no root hairs. had regenerated roots with many root hairs and a shoot (looked like a seedling). 3 20 explants - 1 had callus 20 explants - 2 had callus; no and 4 roots with many root root hairs. hairs. 2. MS+1011M 2,4-D 1 20 explants - none had any 20 explants - none had callus; all callus; 4 explants were were brown. white, the rest brown. 2 20 explants - 2 had possible 20 explants - 2 had callus and 1 callus; 5 explants were of these had some root hairs. white, the rest brown. 3 20 explants - none had 20 explants - 5 had callus and 1 callus; all were brown. of these had root hairs. 3. MS+201.1M 2,4-D 1 20 explants - 1 had callus; 20 explants - 1 had callus and all were brown. appeared to be regenerating a shoot, but too early to tell. 2 20 explants - none had 20 explants - none had callus; all callus; all were brown. were brown or white. 3 20 explants - none had 20 explants - 3 had callus but no callus; all were brown. regeneration; the rest were brown or white.

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Appendix 3.17. Observations of D. tasmanica leaf segment explants grown on 3 different callus-inducing media in the light and the dark for approximately 6 months.

Treatment Rep Light Dark 1. MS+5111■4 2,4-D 1 20 explants - 3 had produced a 20 explants - none had callus; mass of yellow/brown callus all were brown. and roots were regenerating No change. from it. 2 20 explants - 2 had produced 19 explants - 2 had produced yellow/brown callus (as above) a mass of yellow/brown and roots with lots of root callus, but no regeneration hairs were regenerating from from it; 3 were white, the rest it. were brown. 3 20 explants - I had produced 20 explants - 2 had callus; no yellow/brown callus and lots root hairs; 3 were white, the of roots with root hairs were rest were brown. regenerating from it. 2. MS+1011M 2,4-D 1 20 explants - none had any 20 explants - none had callus; callus; 4 explants were white, all were brown. the rest brown. No change. No change. 2 20 explants - 1 had produced a 20 explants - 2 had produced lump of yellow/green callus a large lump of callus and 1 and a green shoot was of these had lot of roots with developing from it. root hairs regenerating from it. 3 20 explants - none had callus; 20 explants - 5 had callus and all were brown and looked 4 of these had produced roots dead. with root hairs; the other had also developed a shoot (looked like a normal seedling). 3. MS+20gM 2,4-D 1 20 explants - 2 had produced 20 explants - 1 had produced lumps of green/brown callus, a lump of callus that was but no regeneration. quite brown; 4 explants were white, the rest brown. 2 20 explants - none had callus; 20 explants - none had callus; 1 was white, the rest brown. all were brown. 3 20 explants - none had callus; 20 explants - 3 had produced 1 was white, the rest brown. a small amount of callus, but no regeneration.

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Appendix 3.18. Observations of M. densiflora whole flower bud explants at 3 different developmental stages, 2 weeks after placement on MS and FBM.

Developmental MS FBM stage 1 8 buds - all had turned brown. 31 buds - 22 remained closed; 2 4 buds remained closed; 3 had had partially opened; 7 had partially opened (1 had a swollen completely opened (1 had a ovary); 1 had completely opened swollen ovary). and the ovary had swollen. 1 - 2 16 buds - 8 remained closed; 5 had 103 buds - 38 remained closed; partially opened; 3 had completely 13 had partially opened; 52 had opened. completely opened (and ovaries 8 were contaminated, were swollen). 24 were contaminated. 4 19 buds - 9 remained closed; 3 had 59 buds - 44 remained closed; 4 partially opened; 7 had completely had partially opened; 11 had opened (1 had a swollen ovary), completely opened.

Appendix 3.19. Observations of M. densiflora whole flower bud explants at 3 different developmental stages, approximately 1 month after placement on MS and FBM.

Developmental MS FBM stage 8 buds - 5 had expanded ovaries (2 31 buds - 26 had globular of these also had expanded swellings at the top of the base structures at the top of the base of of the flower bud (1 also had an the bud); expanded ovary); 1 had a slightly 3 were dead. expanded ovary; 4 were dead (2 of these were also contaminated). 1 - 2 8 buds - 5 remained closed and were 79 buds - 4 remained closed and swollen at the base; 1 was partially had globular swellings at the opened and swollen at the base; 2 base; 13 were partially opened were completely open and ovaries and had swollen ovaries; 30 were were swollen. completely open and had swellings at the base. 19 buds - 1 remained closed and had 59 buds - 1 remained closed and a swollen ovary (and also had was very swollen at the base; 13 swellings at the base); were completely open and had 8 were dead swellings at the base; 10 were contaminated. 45 were contaminated.

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Appendix 3.20. Observations of the remaining M. densiflora whole flower bud explants at 3 different developmental stages, approximately 3 months after placement on MS and FBM.

Developmental MS FBM stage 8 buds - all were dead. 9 buds - 5 were developing green structures (unable to positively identify yet, but possibly shoots). 1 - 2 7 buds - 1 was developing green 20 buds - 2 were developing green structures (possibly shoots). structures (possibly shoots). 4 N/A N/A

Appendix 4.1. Observations of D. latifolia flower bud explants (from each developmental stage) after 2 months growth on FBM.

Developmental Observations stage 1 8 buds - 3 were still green and 1 of these had begun to produce many shoots; 5 had turned brown (1 of these had a swollen ovary). 2 8 buds - 6 were still green but showed no signs of regeneration; 2 were brown and looked dead. 3 7 buds - 3 were still green but showed no signs of regeneration; 4 were brown and looked dead.

Appendix 4.2. Observations of D. latifolia flower bud explants (from each developmental stage) after 4 months growth on FBM.

Developmental Observations stage 1 5 buds - 1 was very green and had produced a mass of shoots; 2 were brown but were starting to produce capsules; 2 were dead. 2 6 buds - 5 were still green and 2 of these had produced 1 shoot each; 1 was dead. 3 3 buds - 2 were still green but showed no signs of regeneration; 1 was dead.

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