The genus Rhododendron (Ericaceae) includes diverse species and phenotypes with a broad range of ornamental characteristics and environmental tolerances. These traits make the genus appealing for breeding novel cultivars for landscape use. Rhododendron ‘Fragrantissimum Improved’ is a unique wide hybrid between R. edgeworthii Hook. and R. formosum Wallich. var. formosum (American Rhododendron Society, 2009) that is an improvement on the leggy and sprawling cultivar R. Fragrantissimum, which has been in the nursery industry for over 100 years. ‘Fragrantissimum Improved’ exhibits a compact growth habit, attractive exfoliating bark, lush evergreen foliage, and clusters of large, white blushed pink, pleasantly fragrant flowers. These ornamental traits are highly desirable for breeding and development of an improved cold-hardy cultivar. Like many wide hybrids, however, R. ‘Fragrantissimum Improved’ is sterile (T.G. Ranney, personal experience).
Hybrid sterility or chromosomal sterility can result from structural differences in chromosomes between species, thus preventing proper alignment during metaphase I of meiosis. This can prevent formation of viable gametes as a result of the presence of univalents and laggard chromosomes (Contreras et al., 2007). Contreras et al. (2007) found laggard chromosomes and bivalent bridges during cell division in the sterile wide hybrid R. ‘Fragrant Affinity’ that also led to infertility. In many cases, fertility can be restored in wide hybrids by doubling the number of chromosomes to produce allotetraploids. Allotetraploids have homologous pairs of chromosomes that allow for disomic pairing and formation of balanced gametes during meiosis (Lu and Bridgen, 1997; Olsen et al., 2006; Ramsey and Schemske, 2002; Ranney, 2006).
Allopolyploids have been successfully induced in many genera, including Buddleia L. (butterfly bush), Lilium L. (lily), Nerine Herb. (cape flower), and Syringa L. (lilac) (Rose et al., 2000a, 2000b; van Tuyl et al., 1992). In addition to restored fertility, allotetraploids often possess improved ornamental characteristics such as thicker, darker-colored leaves, larger flowers, and improved pest or disease resistance, which makes them desirable to breeders (Comai, 2005; Ranney, 2006). Several studies have shown that allotetraploids can be developed successfully in rhododendrons (Jones et al., 2008; Pryor and Frazier, 1968; Sakai et al., 2004). Moreover, the development of allotetraploids of R. ‘Fragrant Affinity’ may restore fertility (Contreras et al., 2007).
In vitro regeneration protocols provide an excellent mechanism for the manipulation of ploidy level, mutation treatment, and transgenic applications. In vitro shoot regeneration protocols have been developed for several Rhododendron species and hybrids belonging to diverse subsections, including R. canadense (L.) Torr. (rhodora), R. mucronulatum Turcz. (Korean rhododendron), R. schlippenbachii Maxim. (royal azalea), R. yedoense var. poukhanense (Lev.) Nakai (Yodogawa azalea), R. ‘Boule de Neige’, and R. ‘Gibraltar’ (McCown and Lloyd, 1982); R. ponticum L. (pontic rhododendron) (Almeida et al., 2005); R. simsii Planch. ‘Helmut Vogel’ (Mertens, 1996); R. catawbiense Michx. ‘English Roseum’ (Sicuranza and Mitkowski, 2007); and R. P.J.M. Group (McCown and Lloyd, 1982; Preece and Imel, 1991). The majority of these species and hybrids belong to the subgenera Hymenanthes, Pentanthera, or Tsutsusi and represent various sections and subsections with only R. mucronulatum and the R. P.J.M. Group representing subgenus Rhododendron (subsections Rhodorastra and Caroliniana) (American Rhododendron Society, 2009). The parents of Rhododendron ‘Fragrantissimum Improved’ are members of subgenus Rhododendron subsection Edgeworthii (R. edgeworthii) and subsection Maddenia (R. formosum var. formosum). No research has been reported on tissue culture protocols for Rhododendron sp. in subsection Edgeworthia or subsection Maddenia.
In vitro shoot regeneration from leaves of Rhododendron sp. is most commonly stimulated by a combination of the cytokinins, 6-(γ,γ-dimethylallylamino) purine (2iP) or zeatin, and auxins, indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). To maximize shoot regeneration from leaves, 2iP and zeatin are typically required in high concentrations often exceeding 50 μM (Iapichino and Chen, 1995; Iapichino et al., 1992; Mertens, 1996; Tomsone and Gertnere, 2003). In comparison, thidiazuron [6-(γ,γ-dimethylallylamino)] (TDZ) has been an effective cytokinin in some species and can be used in lower concentrations, often making it more efficient (Bates et al., 1992; Huetteman and Preece, 1993). Thidiazuron has been used successfully at lower concentrations in several Rhododendron sp. (Preece and Imel, 1991; Samyn et al., 2002). For R. P.J.M. Group, Preece and Imel (1991) found TDZ to be effective at low concentrations with 52 shoots per leaf explant obtained on media supplemented with 10 μM TDZ and 1 μM IBA compared with 21 shoots with 50 μM 2iP and 10 μM IBA.
Efficient in vitro regeneration systems provide an ideal platform for manipulation of ploidy levels. Early polyploidization experiments used colchicine for mitotic inhibition. However, oryzalin is often preferred to colchicine as a result of its reduced toxicity, higher affinity to plant tubulins, effectiveness at lower concentrations, and higher survival of plantlets (Hansen and Anderson, 1996; Sree Ramulu et al., 1991; Väinölä, 2000; van Tuyl et al., 1992). Oryzalin has also been used successfully for in vitro ploidy manipulation in several genera, including Buddleia, Hypericum L. (hypericum), Miscanthus Anderss. (silver grass), and Rosa L. (rose) (Dunn and Lindstrom, 2007; Kermani et al., 2003; Meyer et al., 2009; Petersen et al., 2003; Rose et al., 2000b). However, there have been few studies investigating in vitro ploidy manipulation in Rhododendron. In an intersubgeneric hybrid of an evergreen and deciduous azalea, the highest tetraploid formation (85%) occurred when explants were treated with 300 μM oryzalin for 48 h (Sakai et al., 2004). In another study using one evergreen and two deciduous Rhododendron hybrids, the highest percentage of tetraploids (18%) was formed when explants were treated with 150 μM oryzalin for 24 h (Väinölä, 2000).
Development of an efficient in vitro regeneration system for R. ‘Fragrantissimum Improved’ would provide an avenue for ploidy manipulation for this cultivar and related species. Development of an allopolyploid of this cultivar could further enhance ornamental traits and restore fertility. Thus, the objectives of this research were to 1) develop an efficient methodology for in vitro shoot regeneration from leaves of R. ‘Fragrantissimum Improved’; and 2) develop an oryzalin-mediated protocol for polyploid induction in ‘Fragrantissimum Improved’.
Almeida, R., Gonçalves, S. & Romano, A. 2005 In vitro micropropagation of endangered Rhododendron ponticum L. subsp. baeticum (Bossier and Reuter) Handel-Mazzetti Biodivers. Conserv. 14 1059 1069
American Rhododendron Society: Members worldwide. © 1998–2009 Description of Rhododendron ‘Fragrantissimum Improved’ 4 Nov. 2009 <http://www.rhododendron.org/descriptionH_new.asp?ID=865>.
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