The genus Hypericum L. comprises ≈370 species worldwide that are well known for their pharmaceutical qualities. Several species, including Hypericum frondosum Michx., Hypericum galioides Lam., and Hypericum kalmianum L., also have desirable ornamental characteristics and environmental tolerances that make them promising species for breeding and improvement (Coulter, 1886; Hasselkus, 1998; Touchell et al., 2008). All three species demonstrate broad adaptability and have showy, gold-colored flowers. In addition to attractive flowers, H. frondosum ‘Sunburst’ also has bluish green foliage and a compact growth form. Although generally more open in habit than the other two species, H. kalmianum has the desirable characteristic of being cold-hardy to U.S. Department of Agriculture Zone 4. Hypericum galioides ‘Brodie’ is particularly tolerant of the hot, humid conditions in the southeastern United States. At the North Carolina State Mountain Horticultural Crops Research and Extension Center (MHCREC) in Mills River, NC, these three species have been crossed, through multiple generations, to develop hybrid H2003-004-016, an open-pollinated seedling from hybrid H2001-090 [H. frondosum × (H. galioides ‘Brodie’ × H. kalmianum)]. The hybrid exhibits a dense, compact growth form, narrow, bluish green foliage, and an abundance of showy, gold-colored flowers.
In addition to applications in basic propagation, tissue culture can be a useful tool in improvement of ornamental features. Development of in vitro regeneration systems provides an ideal foundation for further improvements by ploidy manipulations, mutation treatments, and transgenic applications. Previous in vitro regeneration studies of Hypericum have focused on medicinal species, including H. perforatum L. (Franklin and Dias, 2006; McCoy and Camper, 2002; Murch et al., 2000; Pretto and Santarém, 2000), H. heterophyllum Vent. (Ayan and Crak, 2006), and H. brasiliense Choisy (Cardoso and de Oliveira, 1996). Other species have been regenerated in vitro as a conservation effort such as H. foliosum [Dryand.] (Moura, 1998). However, little work has been done to develop in vitro techniques for ornamental Hypericum species with the exception of H. frondosum (Touchell et al., 2008).
In vitro regeneration from leaves of Hypericum sp. characteristically requires a high cytokinin:auxin ratio (Moura, 1998; Santarém and Astarita, 2003). Specifically, work with H. frondosum, a parent of H2003-004-016, shows high levels of benzylamino purine (BA) facilitate optimum in vitro regeneration from leaves (Touchell et al., 2008). Benzylamino purine is the most commonly used cytokinin for in vitro regeneration of Hypericum sp. However, BA has been associated with inducing somaclonal variation (Siragusa et al., 2007) and hyperhydricity (Bairu et al., 2007; Rossetto et al., 1992) in some crops, which may negatively influence the quality of regenerated shoots. In comparison, meta-topolin (mT) is a naturally occurring cytokinin with a similar aromatic structure to BA and has not been linked with somaclonal variation or hyperhydricity (Bairu et al., 2007; Werbrouck et al., 1996), but there is a need for more extensive research of its long- and short-term effects on plant in vitro cultures. Meta-topolin may have potential as a highly active alternative to BA. Although mT has not been used with Hypericum sp., it has shown promise in regeneration of Spathiphyllum floribundum N.E.Br. (Werbrouck et al., 1996), Musa AAB L. (Roels et al., 2005), Aloe polyphylla Schönland ex Pillans (Bairu et al., 2007), and Pelargonium ×hederaefolium ‘Bonete’ Salisb. (Wojtania et al., 2004).
Several auxins have been used in regeneration of Hypericum sp., including indoleacetic acid (IAA) (Ayan and Crak, 2006; Cardoso and de Oliveira, 1996; Franklin and Dias, 2006; Touchell et al., 2008; Wang et al., 2007), α-naphthaleneacetic acid (Cardoso and de Oliveira, 1996; Moura, 1998; Wang et al., 2007), and 2,4-dichlorophenoxyacetic acid (2,4-D) (Ayan and Crak, 2006; Cardoso and de Oliveira, 1996; Pretto and Santarém, 2000; Wang et al., 2007). However, recent work with one of the parents of hybrid H2003-004-016, H. frondosum ‘Sunburst’, has shown 2,4-D to be ineffective for in vitro shoot regeneration (Touchell et al., 2008).
Efficient in vitro regeneration systems provide a tool to manipulate ploidy and improve ornamental features. Polyploidy in some plants may result in desirable ornamental characteristics such as larger, longer-lasting flowers, thicker petals, and larger, thicker leaves (Kehr, 1996). In addition, polyploid plants, particularly allopolyploids, can have other advantageous traits such as enhanced vigor, improved pest resistance and stress tolerance, and protection from deleterious mutations resulting from gene redundancy (Comai, 2005; Ranney, 2006).
Various mitotic inhibitors have been used to induce polyploidy in in vitro systems such as colchicine [N-(5,6,7,9-tetrahydro-1,2,3,10-tetra-methoxy-9-oxobenzo(a)heptalen-7-yl] acetamide. However, the dinitroaniline herbicide oryzalin (3,5-dinitro-N4,N4-dipropylsufanilamide) is often preferred to colchicine for use in polyploidy induction in plants as a result of its lower toxicity (van Tuyl et al., 1992), effectiveness at lower concentrations (Morejohn et al., 1987; Väinölä, 2000; van Tuyl et al., 1992), and tendency to produce plants without deformed tissue or abnormal growth (van Tuyl et al., 1992). For example, in Lilium L., 150 μM oryzalin treatments induced more polyploids than colchicine at 2.5 mm (van Tuyl et al., 1992). Lilium sp. treated with oryzalin also exhibited fewer growth abnormalities than those treated with colchicine. Similarly, oryzalin treatments (30 μM or 150 μM) of Rhododendron L. yielded 45% polyploids as opposed to 15% with colchicine treatments (0.625 mm or 1.25 mm) (Väinölä, 2000). Previous work with polyploid induction in Hypericum is limited to H. perforatum and only uses colchicine (Wang et al., 2007). Therefore, to improve Hypericum H2003-004-016 for release as a new ornamental hybrid and to provide a foundation for future work in in vitro regeneration and ploidy manipulation of Hypericum taxa, the objectives of this study were to 1) develop an effective shoot regeneration protocol for Hypericum H2003-004-016 from leaf explants; and 2) establish a reliable procedure for in vitro induction of polyploidy in this hybrid using oryzalin.
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