Ligustrum Japonicum) Phillips Et Al., 2015; Ranney Et Al., 2007, 2010; Thammina Et Al., 2011; Vining Et Al., Mohammed I
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HORTSCIENCE 51(11):1371–1377. 2016. doi: 10.21273/HORTSCI11138-16 ornamental breeding programs in the United States have initiated breeding projects to develop new cultivars with reduced or elim- Induction, Identification, and inated invasive potential in major invasive ornamental shrubs or trees (Anderson, 2007; Characterization of Tetraploids in Czarnecki et al., 2012; Freyre et al., 2012; Leonhardt and Shi, 2009; Olsen, 2007; Japanese Privet (Ligustrum japonicum) Phillips et al., 2015; Ranney et al., 2007, 2010; Thammina et al., 2011; Vining et al., Mohammed I. Fetouh1 2012). Developing genetic tools and gener- Horticulture Department, Faculty of Agriculture, Tanta University, Tanta, ating germplasm resources that can be used Egypt; and Institute of Food and Agricultural Sciences, Gulf Coast Research for this objective have become essential and important for current and future ornamental and Education Center, University of Florida, 14625 County Road 672, plant breeding (Anderson, 2007; Li et al., Wimauma, FL 33598 2004; Olsen, 2007; Thammina et al., 2011; 2 Vining et al., 2012). Abdul Kareem Several species of Ligustrum L. (privet) Institute of Horticultural Sciences, University of Agriculture, Faisalabad, are commonly used as ornamentals in many Pakistan; and Institute of Food and Agricultural Sciences, Gulf Coast parts of the world (Dirr, 1998; Wilson et al., Research and Education Center, University of Florida, 14625 County 2014). In the landscape, these plants are Road 672, Wimauma, FL 33598 valued for their evergreen leaves, white flowers, adaptability to a range of landscape Gary W. Knox conditions, tolerance to pruning, resistance to Department of Environmental Horticulture, West Florida Research and diseases, and wide availability (Dirr, 1998). Some Ligustrum species have escaped culti- Education Center, Institute of Food and Agricultural Sciences, University of vation and become naturalized in natural Florida, 155 Research Road, Quincy, FL 32351 areas (Munger, 2003). For example, 16 coun- tries report naturalization of chinese privet Sandra B. Wilson (Ligustrum sinense Lour.) (Morris et al.; Department of Environmental Horticulture, Institute of Food and 2002; Munger, 2003). In the United States, Agricultural Sciences, University of Florida, Fifield Hall, Gainesville, FL chinese privet has escaped in 20 states and is 32611 considered invasive in many states (FLEPPC, 2015; Munger, 2003). Glossy privet (Ligus- Zhanao Deng3 trum lucidum W.T. Aiton) has escaped culti- Department of Environmental Horticulture, Gulf Coast Research and vationin10statesintheUnitedStates Education Center, Institute of Food and Agricultural Sciences, University (Munger, 2003; Wilson et al., 2014) and is also a weed in Australia (Panetta, 2000), New of Florida, 14625 County Road 672, Wimauma, FL 33598 Zealand (Miller and Henzell, 2000), and Additional index words. chromosome doubling, colchicine, DNA content, indole butyric acid, Argentina (Hoyos et al., 2010). Japanese privet polyploid, ploidy manipulation, flow cytometry (L. japonicum Thunb.) is native to Japan and eastern Asia and was introduced to the United Abstract. A number of privet species (Ligustrum spp.) that are important to the nursery States from Japan and Korea in 1845 as an and landscape industry have escaped cultivation and become invasive or weedy in the ornamental landscape plant (Munger, 2003; United States and other countries. Induced tetraploids in these species may produce new Wilson et al., 2014). Since then, this species selections or cultivars with reduced or eliminated invasive potential. Applying drops of hasbeenwidelyusedinthelandscapeinthe semisolid agar containing 0.1% to 0.3% colchicine and 0.2% dimethyl sulfoxide (DMSO) southeastern, southern, and western United to newly emerged seedlings of japanese privet (Ligustrum japonicum Thunb.) resulted in States. It has escaped cultivation and become 15.6% to 22.6% tetraploid induction. The nuclear DNA content of tetraploids was 5.31 naturalized in 12 southeastern states in the pg/2C, 101.9% higher than that of diploids. Compared with diploid plants, tetraploids United States (Munger, 2003). Japanese privet were more compact, with an average of 31.0% shorter plant height and 33.1% smaller commonly forms dense thickets in the field or canopy width. Tetraploids had 29.2% thicker internodes, and their leaves were 39.5% forest understories, shading and displacing larger and 33.8% thicker, resulting in 42.1% to 24.1% greater fresh or dry leaf weights many native species. Once established, it is (per leaf) in tetraploids compared with diploids. Without indole-3-butyric acid (IBA) difficult to eradicate. treatment, cuttings from tetraploids showed 28% lower rooting than diploids. IBA The invasiveness of many ornamentals is treatments improved the rooting of tetraploid cuttings, resulting in 65% rooting success. attributed to a number of factors including These results indicate that tetraploids can be readily induced in japanese privet and prolific production of viable seeds. Thus, induced tetraploids show significant changes in plant growth and size, shoot growth, leaf making plants sterile or seedless can reduce, morphology, and rooting of cuttings. The modified tetraploid induction method and the even eliminate, their invasive potential induced tetraploids are expected to be useful for producing new selections or cultivars (Anderson, 2007; Ranney, 2004, 2006). Sev- with reduced invasive potential in japanese and other privets. eral genetic tools have been used to reduce seed production, viability, and/or germi- nation, including natural mutations, artifi- Exotic plant invasions are considered plant species in the United States are esti- cial mutagenesis, interspecific hybridization, one of the main causes of the degradation mated at nearly $35 billion (Pimentel et al., ploidy manipulation (Czarnecki et al., 2012; of ecosystems and the loss of biodiversity 2005). To help mitigate this huge impact and Freyre et al., 2012; Trueblood et al., 2010), globally (Theoharides and Dukes, 2007). meet the nursery and landscape industry’s endosperm culture (Thammina et al., 2011), Ornamental horticulture has been recog- need for plant materials, horticulturists have and transgenics (Li et al., 2004; Vining et al., nized as the main source of plant invaders been searching for cultivars with reduced 2012). So far, ploidy manipulation has resulted (Bell et al., 2003; Niemiera and Von Holle, invasive potential (Knox and Wilson, 2006; in considerable success and yielded multiple 2009; Reichard and White, 2001; Rejmanek, Trueblood et al., 2010; Wilson and Mecca, sterile, noninvasive cultivars or breeding lines 2014). The economic impacts of invasive 2003; Wilson et al., 2004, 2012). Multiple in several important ornamental plants, such as HORTSCIENCE VOL. 51(11) NOVEMBER 2016 1371 Hypericum androsaemum L. (Trueblood et al., a model to evaluate the effectiveness and and Deng (2009). The CyStain Ultraviolet 2010), Lantana camara L. (Czarnecki et al., efficiency of the semisolid agar method for Ploidy Precise P dye (Partec) was amended 2012), and Ruellia simplex Wright (Freyre induction of stable tetraploids in privet and to with 1.25% (w/v) polyvinylpyrrolidone (PVP) et al., 2012). characterize the effects of chromosome dou- and 0.125% (v/v) b-mercaptoethanol (ME) to The key step in ploidy manipulation is bling and tetraploidy on shoot growth and improve the sharpness of the peaks indicating chromosome doubling and induction of sta- leaf morphology. ploidy levels. Nontreated seedlings (controls) ble tetraploids, which are the gateway to were used as the diploid reference. Identified obtain other ploidy levels (triploids, penta- Materials and Methods tetraploids were confirmed by analyzing ad- ploids, hexaploids, octoploids, etc.) through ditional leaves on 4-year-old plants in Mar. interploid crosses. Several antimitotic agents Plant materials. Open-pollinated seeds were 2015 using a CyFlowÒ Cube 6 flow cytometer have been used to inhibit the separation of collected from mature shrubs of L. japonicum (see below). chromosomes at the anaphase of cell division ‘Texanum’ grown at the University of Florida Nuclear DNA content determination and and achieve chromosome doubling in orna- (UF) North Florida Research and Education ploidy confirmation. Nuclear DNA contents mental plants (Contreras, 2012; Contreras Center, Quincy, FL, in Feb. 2010 and of identified tetraploid plants were deter- et al., 2010; Nadler et al., 2012; Vining shipped to the UF Gulf Coast Research and mined on a CyFlowÒ Cube 6 flow cytometer et al., 2012). Colchicine has been one of the Education Center (GCREC), Wimauma, FL. (Sysmex; Partec GmbH Otto-Hahn-Strasse widely used agents to induce tetraploids in Dry seeds were sown onto a commercial 32 D-48161, Munster,€ Germany) using the numerous ornamental plants (Abdoli et al., potting mix (Metro Mix 200; Sun Gro Horti- procedure described by Dolezel et al. (2007) 2013; Henny et al., 2009; Lehrer et al., 2008; culture, Agawam, MA) in plastic containers and modified by Cao et al. (2014). Four plants Leonhardt and Shi, 2009). Several types of (15 cm in diameter) in Feb. 2011. Seeds were with known nuclear DNA contents (Dolezel plant tissues or organs, such as seeds, seed- covered with a layer of horticultural grade et al., 2007) were tested as potential internal lings, axillary buds or shoot tips, embryos, vermiculite (1 cm thick) and germinated in references. Radish (Raphanus sativus ‘Saxa’) and cultured cells or tissues, have been used a growth