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; Rejm anek, 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 Dole zel 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 (Dole zel 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 room at 24 Cwith16hoflight with a nuclear content of 1.11 pg/2C was as the target for colchicine treatment with (120 to 150 mmol·m–2·s–1)and8hofdark. selected as the internal reference for use in various rates of successful tetraploid induc- About 5 weeks after sowing, seedlings emerged this study. Using a sharp razor blade, fresh tion and mixoploidy (Habbard et al., 2016; with fully expanded cotyledons. tender leaves were cochopped with a similar Henny et al., 2009; Jones et al., 2007; Lehrer Induction, identification, and confirmation amount of leaf tissue of radish in 1 mL of cold et al., 2008; Leonhardt and Shi, 2009; Vining of tetraploids. The semisolid agar method of LB01 lysis buffer to release the nuclei. The et al., 2012). The availability of an effective Jones et al. (2007) was used with some lysis buffer contained 15 mM Tris, 2 mM and efficient chromosome doubling and tet- modifications. Colchicine was applied to the Na2EDTA, 0.5 mM spermine tetrahydrochlo- raploid induction protocol is critical for growing points of newly emerged seedlings at ride, 80 mM KCl, 20 mM NaCl, and 0.1% (v/v) successful ploidy manipulation and develop- the cotyledonary stage to induce tetraploids. Triton X-100. The buffer was adjusted to pH ment of sterile, noninvasive cultivars in in- A 1% colchicine stock solution was made by 7.5 and filter-sterilized. Before use, ME was vasive ornamental plants. dissolving colchicine power (Sigma-Aldrich, added to the lysis buffer to a final concentra- Our literature searches and quick surveys St. Louis, MO) in water. The colchicine stock tion of 15 mM. The collected nuclei (in 1mL of privet cultivars indicated a lack of tetra- solution was added to melted semisolid agar of lysis buffer) were stained with 50 mLof ploids in glossy, chinese, and japanese privet. containing one-fourth-strength Murashige and DNA fluorochrome propidium iodide stock Preliminary work on treating axillary buds of Skoog (MS) basal salts, 0.55% agar, and DMSO solution (1 mg·mL–1) (Sigma-Aldrich) and privet plants with colchicine resulted in few (Sigma-Aldrich; final concentration 0.2%) to 50 mL of RNase stock solution (1 mg·mL–1) solid tetraploids. Thus, the main objectives a final concentration. Three colchicine concen- (Sigma-Aldrich) for 2 to 5 min at ambient of this study were to use japanese privet as trations were used: 0.1%, 0.2%, and 0.3%. The temperatures and in dark conditions. The colchicine-containing agar solution was kept at stained nuclei suspension was then analyzed 40 C and then 30 mL were pipetted onto the by flow cytometry. At least three runs were growing point of each privet seedling. Treat- performed for each privet sample, and at Received for publication 7 July 2016. Accepted for ed seedlings were placed in a high humidity least 3000 nuclei were counted in each run. publication 29 Aug. 2016. (100% relative humidity) growth chamber at The nuclear DNA content of the japanese This project was funded in part by USDA-NIFA hatch 24 C in dark to minimize colchicine degra- privet samples was calculated according to projects FLA-GCR-005065 and FLA-GCC-005605, dation by light. Control seedlings received Dole zeletal.(2007):samplenuclearDNA and a grant from the Horticultural Research Institute semisolid agar without colchicine. All treat- content (pg/2C) = internal reference nuclear (project no. 1390-696 to C.R. Adams and Z. Deng). · We thank Gail Bowman and Joyce Jones for their ments were repeated during three consecu- DNA content (1.11) (mean fluorescence technical assistance and Zhe Cao for assistance with tive days. value of sample/mean fluorescence value of flow cytometry analysis. We thank Jaroslav Dole zel After treatment, seedlings were grown in internal reference). (Laboratory of Molecular Cytogenetics and Cytome- a greenhouse, and fertigated with 50 to 100 Morphological characterization. Four tet- try, Institute of Experimental Botany, Olomouc, Czech ppm nitrogen as needed. Seedlings were kept raploid and four diploid plants (4 year old) Republic) for providing seeds of the internal standards in community containers until they produced were selected in Feb. 2015 for morphological for flow cytometry analysis. We are grateful to M.E. shoots with several true leaves and later trans- characterization. Plant heights were measured EL-Denary, Carrie Reinhardt Adams, and Rosanna planted individually into new containers (10 to from the ground level vertically to the highest Freyre for reviewing this manuscript and providing 15 cm in diameter). Seedlings were grown in point of the plant canopy; plant widths were valuable comments. Mohammed I. Fetouh confirmed the ploidy levels, these containers for 1year,andthentrans- measured by projecting the canopy of individ- determined nuclear DNA contents, characterized plant planted into ground beds at the UF’s GCREC ual plants to the ground and measuring the morphological changes and growth characteristics, in Mar. 2013. All transplanted plants were widths of the projected area on the ground in conducted rooting experiments, collected and ana- grown in full sun, irrigated through drip tapes, two perpendicular directions. Four straight lyzed data, prepared figures, and drafted the manu- and fertilized with 15 g of 15N–3.9P–12K terminal shoots (40 cm in length) on each script. Abdul Kareem germinated seeds and treated controlled-release fertilizer (Osmocote; Scotts, of the selected tetraploid and diploid plants seedlings to induce tetraploids. Gary W. Knox and Marysville, OH). were selected for determining shoot charac- Sandra B. Wilson grew parental plants, collected and To identify tetraploids, multiple fully ex- teristics. The selected shoots were cut off the processed seeds. Zhanao Deng designed the experi- ments, supervised the project, reviewed experiments panded young leaves were collected from each plants and all leaves were removed from the and data, and revised and finalized the manuscript. plant, and each leaf was analyzed separately shoots at the base of the leaves. The bare 1Visiting scholar. for ploidy level in Jan. and Feb. 2013. The shoots were used to measure the internode 2Visiting graduate student. Partec PA Ploidy Analyzer (Partec, Munster,€ length, internode diameter, and shoot fresh 3Corresponding author. E-mail: zdeng@ufl.edu. Germany) was used as described by Czarnecki weight. The shoots were then oven dried at
1372 HORTSCIENCE VOL. 51(11) NOVEMBER 2016 70 C to a constant mass before shoot dry RSERs were calculated according to Morris Rooting of tetraploid cuttings and effect of weight determination. et al. (2002). IBA concentration. Semi-hardwood terminal To measure relative stem elongation log L2 – log L1 shoots, 10 cm long and consisting of four to rates (RSER), branches with expanding leaf RSER = e e five nodes, were taken from tetraploid and t2–t1 buds (1–2 cm) were selected at random diploid plants on 1 Mar. 2015 and brought and labeled in Mar. 2015. The lengths of the where L1 is the shoot length at the start of the back to the laboratory. Shoots were re-cut at selected branches were measured weekly time interval t1, L2 is the shoot length at the 0.5 cm below the third node (from tip) at for 5 weeks during March and Apr. 2015 end of the time interval t2, and RSER is a45 angle. Then, leaves at the lower two from the bud scale scars to the tip of the expressed as cm·week–1. nodes were removed, and the leaf at the branches when the shoots were elongating. To determine leaf characteristics, mature apical tip was kept. Prepared cuttings were leaves were harvested from each of the ran- arranged in groups of 40 with a rubber band domly selected stems, and the number, area, and completely soaked in water for 24 h to thickness, and fresh weight of leaves were leach out potential rooting inhibitors (Wang recorded. The harvested leaves were then oven and Zhong, 2012). Soaked cuttings were then dried at 70 C to a constant mass to obtain dry briefly immersed in a fungicide solution weights. Leaf areas were measured according (Banrot 40WP, Everris NA, Inc., Dublin, to Matthew et al. (2002); digital images of OH; 0.53 g·L–1) followed by a 5-s dip of their individual leaves were obtained from a flatbed lower ends in a root-promoting solution to scanner at a resolution of 300 dots per inch a depth of 2 cm. Three different concentra- and then their area was determined using tions of IBA were used (1000, 2500, or 5000 Scion Image (version 4.0.2; Scion Corp., mg·L–1) for the root-promoting solution. The Frederick, MA). Leaf area and dry mass data control was water without any IBA. Cuttings were used to derive the specific leaf area were then stuck into 40-cell propagating trays (SLA)andspecificleafmass(SLM).SLA (cells 3 · 3 · 3 cm) filled with potting mix was calculated according to Cornelissen (FafardÒ 3B; Sun Gro Horticulture). Trays et al. (2003): leaf area was divided by the were placed on a metal bench in a glasshouse Fig. 1. Seedlings of ‘Texanum’ japanese privet dry mass of the leaves and expressed in under an automatic mist system run for 3 s at shortly after their growing points between mm2·mg–1. SLM was calculated according to 5-min intervals during the day. Air tempera- cotyledons were exposed to colchicine (final concentration 0.1% to 0.3%) in droplets of Larcher (2003): leaf dry mass was divided tures in the glasshouse were 18 to 28 C, relative semisolid agar containing 0.2% dimethyl sulf- by the one-side area of fresh leaves and humidity was 70% to 90%. After 8 weeks, –2 oxide and one-fourth-strength Murashige and expressed in mg·mm (thus SLM is the cuttings were harvested and the number and Skoog basal salts. inverse of SLA). fresh weight of roots on each cutting, the length (cm) and diameter (mm) of the longest root on each cutting, and the percentage of cuttings Table 1. Effect of colchicine concentrations on seedling survival and tetraploid induction in japanese privet rooted were recorded. The rooting experiment (Ligustrum japonicum). was repeated in May 2015. Colchicine Individuals Survival Tetraploids Avg tetraploid Mixoploids, Statistical analysis. Plant, shoot, and leaf concn (%) treated (no.) (%) (no.) induction rate ± SD (%) no. (%) morphological data were analyzed using a ran- Control 10 100.0 0 0 0 domized complete block design with the ploidy 0.1 67 64.2 9 20.9 ± 6.5 8 (11.9) level as the main factor. The rooting experi- 0.2 49 71.4 10 22.6 ± 12.0 8 (16.3) ment was analyzed using a completely ran- 0.3 66 68.2 7 15.6 ± 10.6 7 (10.6) domized design with ploidy level as factor A
Fig. 2. Flow cytometry histograms of (A) a typical diploid and (B) an introduced tetraploid) of japanese privet generated by a CyFlowÒ Cube 6 flow cytometer. Diploid and tetraploid leaf cells were stained with propidium iodide and the relative fluorescence of individual nuclei was observed on a CyFlowÒ Cube 6. The horizontal axes (Channels FL2) show the relative fluorescence of individual nuclei and the vertical axes show the number of nuclei.
HORTSCIENCE VOL. 51(11) NOVEMBER 2016 1373 and IBA concentration as factor B. The rooting Table 2. Nuclear DNA contents of japanese privet (Ligustrum japonicum) diploids and induced tetraploids experiment was replicated four times and with determined by flow cytometry. 10 cuttings treated per combination of ploidy Ploidy Individuals Nuclear DNA Mean nuclear DNA level and IBA concentration. The experiment level analyzed (no.) contents (pg/2C) content ± SD (pg/2C) was repeated once and similar results were 2x 7 2.52–2.77 2.63 ± 0.06 obtained. Thus, both results were combined 4x 5 5.07–5.43 5.31 ± 0.13 and analyzed using the MSTAT-C program (Michigan State Univ., East Lansing, MI) for variance and mean differences (least significant Table 3. Morphological comparison between diploid and induced tetraploid plants of japanese privet difference test at P = 0.05). (Ligustrum japonicum). Samples Tetraploids (percent Results analyzed (no.) Traits Diploids change over diploids) Trees 8 Plant height (cm) 155.63 107.33 (–31.0%)* Induction of tetraploids. Seeds of ‘Texanum’ 8 Canopy width (cm) 122.00 81.67 (–33.1%) japanese privet germinated readily with an Shoots 60 Internode length (cm) 2.52 3.17 (+25.8%)NS average of 42% emergence within 5 weeks. 60 Internode diameter (mm) 3.57 4.62 (+29.3%)* By this time, the majority of seedlings (80%) 60 Fresh weight (g) 6.95 7.20 (+3.6%)NS NS had two cotyledons with a very small growing 60 Dry weight (g) 3.49 3.59 (+3.0%) · –1 NS point in between them, which is the ideal stage 32 RSER (cm week ) 0.44 0.32 (–27.7%) Leaves 240 Leaves per shoot (40 cm long) 32.50 25.25 (–22.3%)NS for the semisolid agar method. After three 240 Leaf area (cm2) 12.81 17.88 (+39.5%)* applications of the semisolid agar-containing 240 Leaf thickness (mm) 0.62 0.82 (+33.8%)* colchicine, the growth of some seedlings was 240 Fresh leaf weight (g) 0.78 1.11 (+42.1%)* inhibited, but others resumed growth and pro- 240 Dry leaf weight (g) 0.28 0.35 (+24.1%)* duced shoots (Fig. 1). Multiple leaves were 240 Specific leaf area (mm2·mg–1) 45.43 51.19 (+12.7%)NS collected from each of the treated seedlings for 240 Specific leaf mass (mg·mm–2) 0.02 0.02 (0.0%)NS ploidy analysis. On average, 16% to 23% of RSER = relative stem elongation rate (cm·week–1). treated seedlings were solid tetraploids, and NS, *Nonsignificant or significant at P # 0.05, respectively. 11% to 16% were mixoploids containing diploid and tetraploid cells, or tetraploid and octoploid cells in the same plants (Table 1). 42.1% and 24.1% greater than the fresh and dry Mixoploid plants were discarded before trans- leaf weight of diploids, respectively. plant to the ground beds. Two years after the Rooting of tetraploid cuttings. When IBA initial analysis, all solid tetraploids were con- was not applied to cuttings, 45.0% of the firmed by analyzing the ploidy levels of leaves tetraploid cuttings rooted within 8 weeks after from multiple new shoots. There were no they were stuck into the rooting substrate, significant differences among the three col- whereas 62.5% of the diploid cuttings rooted chicine concentrations used (0.1%, 0.2%, (Fig. 4A). Tetraploid cuttings appeared to and 0.3%) in terms of survival rate, tetraploid exhibit 26.5% to 35.5% lower rooting rates than induction rate, and frequency of mixoploids diploid cuttings when IBA was applied at 1000, (Table 1). 2500, or 5000 ppm, although the differences Nuclear DNA contents. Sharp histograms between diploid and tetraploid cuttings in root- of relative fluorescence and small coefficients ing percentage were not statistically significant. of variation were obtained for the samples When IBA was not applied, tetraploid analyzed (Fig. 2). Based on ‘Saxa’ radish as an cuttings produced new roots of 40.2% greater Fig. 3. Typical leaves and transverse sections of internal reference, the average nuclear DNA diameter or thickness than the roots produced (A) diploid and (B) tetraploid japanese privet contents of diploid and tetraploid japanese by diploid cuttings (Fig. 4D and F). Diploid plants showing changes in leaf size and thickness. privet were 2.63 and 5.31 pg/2C, respectively cuttings developed thicker roots when IBA (Table 2). Induced tetraploids contained 102% was applied and the diameter of new roots more nuclear DNA than the diploids. appeared to increase with the increase of IBA Morphological comparisons. Significant concentration. On the other hand, the diameter within several months using colchi- differences were observed between tetraploid of roots from tetraploid cuttings did not change cine. Newly emerged privet seedlings each and diploid japanese privet seedling in a number significantly with the application of IBA or the had a growing point between two relatively of morphological characters (Table 3). The most change of IBA concentration. Thus, when 2500 large cotyledons. This structure allowed for obvious difference was the smaller stature of the and 5000 ppm of IBA were applied, tetraploid easy placement of droplets of colchicine- induced tetraploids. On average, the induced and diploid cuttings did not show any significant containing semisolid agar onto the growing tetraploid plants were 31.0% shorter and their differences in root diameter. point, and privet seedlings were tolerant of canopy width was 33.1% smaller than the In terms of the number, length, and fresh the applied colchicine. This method has been diploid plants. The internodes of the tetraploids weight of roots per cutting, tetraploid and applied to two other privet species, and dozens were significantly thicker (29.3% increase). diploid cuttings did not show significant more tetraploids have been obtained in these Tetraploids and diploids had similar fresh and differences when IBA was not applied species (Fetouh and Deng, unpublished data). dry shoot weights. During the growing season, (Fig. 4B, C, and E). IBA treatments increased The availability of these tetraploids will make the shoots of tetraploids and diploids elongated the root length of diploid cuttings (2500 and it possible to create other polyploidy levels by an average of 0.32 to 0.44 cm per week. 5000 ppm) and the fresh root weight of in privets. Multiple polyploidy levels will be Leaves of tetraploids were much larger and tetraploid cuttings (Fig. 4C). valuable for developing new privet cultivars thicker, with 39.5 and 33.8% increase in leaf or breeding lines with sterility and reduced area and leaf thickness, respectively, compared Discussion invasive potential and for understanding the with the leaves of diploids (Fig. 3; Table 1). effects of polyploidy on privet growth, devel- These larger and thicker leaves also resulted in This study reveals that the semisolid agar opment, and fecundity. greater leaf fresh and dry weights in tetraploids. method of Jones et al. (2007) can be readily Two modifications were made to the origi- The average fresh and dry weight of individual applied to japanese privet and relatively high nal semisolid agar method of Jones et al. (2008), leaves were 1.11 and 0.35 g in tetraploids, frequencies of solid tetraploids can be induced including 0.2% DMSO and one-fourth-strength
1374 HORTSCIENCE VOL. 51(11) NOVEMBER 2016 Fig. 4. Rooting and root characteristics of diploid and tetraploid cuttings with and without indole butyric acid (IBA) application. (A) Rooting percent, (B) number of roots per cutting, (C) total length of all roots per cutting (cm), (D) diameter of roots (mm), and (E) the fresh weight of all roots per cutting (g). Boxes show the interquartile ranges including 25% to 75% of the data, and squares indicate the mean values of the data. Boxes headed by different letters differ significantly for each other according to the least significant difference at P = 0.05. (F) Roots generated from diploid (left) and tetraploid cuttings (right).
MS basal salt mixture. DMSO has been targeted tissues and cells and increase the salts added in the semiagar was intended to commonly used to facilitate the penetration effectiveness of colchicine treatments (Sanders provide some nutrients to promote cell division of chemical mutagens such as colchicine into and Hull, 1970). The one-fourth-strength MS in the growing point. However, specific effects
HORTSCIENCE VOL. 51(11) NOVEMBER 2016 1375 of these modifications remain to be determined similar to morphological changes observed in H.T. Steege, M.G.A. Van der Heijden, J.G. for the induction of tetraploids in privets. In induced autotetraploids in several other woody Pausas, and H. Poorter. 2003. A handbook of tetraploid induction experiments, the concen- plants including crape myrtle (Lagerstroemia protocols for standarised and easy measure- tration of colchicine was considered to be indica L.), rose (Rosa L.), and London plane ment of plant functional traits worldwide. critical. Up to 2% colchicine was used in [Platanus acerifolia (Aiton) Willd.] (Kermani Austral. J. Bot. 55:335–380. Czarnecki, D.M., II, S.B. Wilson, G.W. Knox, R. previous reported experiments (Habbard et al., 2003; Liu et al., 2007; Ye et al.; 2010). Freyre, and Z. Deng. 2012. UF-T3 and UF-T4: et al., 2016). Higher concentrations of colchi- With these morphological changes, japanese Two sterile Lantana camara cultivars. Hort- cine resulted in high mortalities of treated privet tetraploid plants seemed to have higher Science 47:132–137. seedlings, buds, or shoots, and deformed new ornamental values. Studies have been initiated Czarnecki, D.M., II and Z. Deng. 2009. Occurrence growth, and/or high frequencies of mixoploids to further assess the beneficial effects of tetra- of unreduced female gametes leads to sexual (Henny et al., 2009; Thao et al., 2003). In this ploidy on japanese plant performance and polyploidization in Lantana. J. Amer. Soc. Hort. study, the three concentrations of colchicine tolerance to severe pruning in the landscape. Sci. 134:560–566. (0.1%, 0.2%, and 0.3%) used were equally Several studies have shown that autotetraploids Dirr, M.A. 1998. Manual of woody landscape effective and resulted in similar tetraploid tend to exhibit better tolerance to droughts or plants. Their identification, ornamental charac- induction rates as well seedling survival and water deficits (Allario et al., 2013; Liu et al., teristics, culture, propagation and uses. 5th ed. Stipes Publ., Champaign, IL. mixoploids. To minimize the quantity of col- 2011). It will be of interest to explore whether Dole zel, J., J. Greilhuber, and J. Suda. 2007. Esti- chicine needed, we recommend the use of 0.1% these japanese privet tetraploids show any in- mation of nuclear DNA content in plants using colchicine and three consecutive applications crease in drought tolerance. flow cytometry. Nat. Protoc. 2:2233–2244. to young (5-week old) privet seedlings over It is expected that future sterile polyploid FLEPPC (Florida Exotic Pest Plant Council). 2015. aperiodof3d. privet cultivars or breeding lines with re- Florida Exotic Pest Plant Council’s 2015 list Accurate, efficient ploidy analysis is es- duced or eliminated invasive potential will of invasive plant species. 31 Jan. 2016. sential for rapid identification of polyploids depend on cuttings for commercial propaga-
1376 HORTSCIENCE VOL. 51(11) NOVEMBER 2016 tools to neutralize their invasiveness. Crit. Rev. Panetta, F.D. 2000. Fates of fruits and seeds of Thao, N.T.P., K. Ureshino, I. Miyajima, Y. Ozaki, Plant Sci. 23:381–389. Ligustrum lucidum W.T. Ait. and L. sinense and H. Okubo. 2003. Induction of tetraploids in Liu, S., S. Chen, Y. Chen, Z. Guan, D. Yin, and Lour. maintained under natural rainfall or ornamental Alocasia through colchicine and F. Chen. 2011. In vitro induced tetraploid of irrigation. Austral. J. Bot. 48:701–705. oryzalin treatments. Plant Cell Tissue Organ Dendranthema nankingense (Nakai) Tzvel. shows Phillips, W.D., T.G. Ranney, D.H. Touchell, and Cult. 72:19–25. an improved level of abiotic stress tolerance. T.A. Eaker. 2015. Developing non-invasive Theoharides, K.A. and J.S. Dukes. 2007. Plant Scientia Hort. 127:411–419. callery pears: Fertility and reproductive bi- invasion across space and time: Factors Liu, G., Z. Li, and M. Bao. 2007. Colchicine- ology of triploid cytotypes. SNA Res. Conf. 60th affecting nonindigenous species success dur- induced chromosome doubling in Platanus Annu. Rpt. 60:205–208. ing four stages of invasion. New Phytol. acerifolia and its effect on plant morphology. Pimentel, D., R. Zuniga, and D. Morrison. 2005. 176:256–273. Euphytica 157:145–154. Update on the environmental and economic Trueblood, C.E., T.G. Ranney, P.L. Nathan, J.C. Matthew, E.O., A.L. Douglas, and R. Isaacs. 2002. costs associated with alien-invasive species in Neal, and R.T. Olsen. 2010. Evaluating fertility An inexpensive accurate method for measuring the United States. Ecol. Econ. 52:273–288. of triploid clones of Hypericum androsaemum leaf area and defoliation through digital image Ranney, T.G. 2004. Population control: Develop- L. for use as non-invasive landscape plants. analysis. J. Econ. Entomol. 95:1190–1194. ing non-invasive nursery crops. Combined HortScience 45:1026–1028. Miller, S.A. and R.F. Henzell. 2000. Seed germi- Proc. Intl. Plant Propagators’ Soc. 54:604–607. Vining, K.J., R.N. Contreras, M. Ranik, and S.H. nation of weeds from New Zealand native plant Ranney, T.G. 2006. Polyploidy: From evolution to Strauss. 2012. Genetic methods for mitigating communities. Agron. Soc. N.Z. 12:115–116. new plant development. Combined Proc. Intl. invasiveness of woody ornamental plants: Re- Missouri Botanical Garden. 2016. IPCN chromo- Plant Propagators’ Soc. 56:137–142. search needs and opportunities. HortScience some reports. 25 Aug. 2016.
HORTSCIENCE VOL. 51(11) NOVEMBER 2016 1377