many invasive trees is attributed to Polyploidy as a Management Strategy for their seed fecundity (Pheloung et al., Invasive Species 1999; Wickert et al., 2017). Some of the most invasive trees, such as mico- nia (Miconia calvescens) are invasive Kenneth W. Leonhardt1 primarily because of their prolific seed production. A single 10-m tree with 100 inflorescences, 300 fruit/ ADDITIONAL INDEX WORDS. Cassia, Delonix, induced sterility, Spathodea, Albizia inflorescence, and 100 seeds/fruit, SUMMARY. Most invasive species are prolific seed-producing landscape ornamental will produce 3 million seeds two to plants that have been introduced to non-native habitats with limited or no natural threetimesperyear(Medeirosand controls on their reproduction and spread. Techniques for converting prolific seed- Loope, 1997). producing landscape ornamentals into sterile or nearly sterile forms are available. The distribution of ornamentals Oryzalin and colchicine have been used to double chromosomes, resulting in au- in horticulture is a familiar mecha- totetraploids with reduced fertility and potential parent plants of sterile triploids. Guard cell measurements and flow cytometry have been used to determine ploidy nism of an invasive species’ introduc- conversion and identify polyploids. Complete sterility has been achieved in three tion (Dehnen-Schmutz et al., 2007; species of shower trees (Cassia sp.), and up to a 95% reduction in seed production Pemberton and Liu, 2009; Reichard has been achieved in royal poinciana (Delonix regia) and african tulip tree (Spathodea and White, 2001). Inducing sterility campanulata). Monkey pod (Albizia saman) crosses have produced triploid prog- could prevent otherwise invasive spe- eny to evaluate for sterility. cies from taking hold in non-native ecosystems. Creating tetraploid forms ith human movement comes harm to human health’’ (U.S. Depart- of potentially invasive ornamentals thearrivalofinvasivespecies. ment of Agriculture, n.d.). offers two opportunities to induce WSince humans started migrat- Invasive species can spread easily sterility by altering the mechanism ing to new places, new plants intention- and cause significant ecological dam- for seed production. Because repro- ally came with them for agricultural or age (Blackburn et al., 2011; Pysek and duction is an essential component of ornamental purposes. New plants also Richardson, 2010; Vilaetal.,2011). the stages in which an introduced came unintentionally, via seeds that They are also responsible for great species becomes invasive (Blackburn hitchhiked on clothing, animals, and economic loss—an estimated $120 et al., 2011), inhibiting seed produc- ships. The advent of widespread human billion in 2004 (Pimentel et al., tion is an effective strategy to arrest movement ushered collateral damage 2005). Few comprehensive studies re- the invasiveness of an introduced spe- from a previously unconsidered source: lated to the economic impact of in- cies. Tetraploid forms of ornamentals invasive species. The U.S. Department vasive species have been published are sometimes sterile (Leonhardt, of Agriculture identifies invasive spe- since that of Pimentel et al. (2005), 2016, 2017b) or have highly reduced cies by two key criteria: 1) that it is a but it is recognized that invasive spe- seed production capability (Leonhardt non-native species and 2) that its intro- cies continue to have an impact on and Shi, 2009). If the tetraploid form duction ‘‘causes or is likely to cause local ecosystems’ endangered species. is fertile, it can be crossed with a diploid economic or environmental harm or Invasive plant species are detrimental form of the same species (Vining et al., to 42% of endangered species in the 2012). A successful cross between United States and 18% of endangered tetraploid and diploid plants results Received for publication 25 Feb. 2019. Accepted for publication 11 July 2019. species in the United States have in- in triploid progeny. Most triploids Published online 27 August 2019. vasive plant species as the primary are highly sterile (Ranney, 2000; Vin- cause of their decline (U.S. Depart- ingetal.,2012). 1Department of Tropical Plant and Soil Sciences, University of Hawaii, 3190 Maile Way, St. John ment of Agriculture, n.d.). Polyploidy does not ensure ste- 102, Honolulu, HI 96822-2279 Invasive species are clearly a prob- rility. There are many examples of This work was supported by the U.S. Department of lem and one of the key parameters highly fertile polyploidy species that Agriculture National Institute of Food and Agricul- used to classify them as invasive is have become invasive. For example, ture, Hatch project 819, managed by the College of Tropical Agriculture and Human Resources, Univer- their seed production. High seed the tetraploid form of purple loose- sity of Hawaii. production compared with native strife, Lythrum salicaria, is invasive in The assistance of Dr. Karen Selph, Specialist and speciesisoneoftheparametersthat 13 states of the United States and Graduate Faculty, Department of Oceanography, makes an introduced species invasive provinces of Canada (Kubatova et al., and Director of the School of Ocean and Earth Science and Technology Flow Cytometry Facility, (James et al., 2010). The success of 2008) and the tetraploid form of reed University of Hawaii at Manoa, is greatly appreciated. This paper was part of the Invasive Plants Research Professional Interest Group workshops ‘‘Strategies for Mitigating Invasiveness of Native Species,’’ held 30 Units Sept. 2017 in Waikoloa, HI, and ‘‘It’s Native. Wait! To convert U.S. to SI, To convert SI to U.S., It’s Exotic . . . . Oh No, It’s a Nuisance!’’ held 3 Aug. multiply by U.S. unit SI unit multiply by 2018 in Washington, DC. 29,574 fl oz mL 3.3814 · 10–5 K.W.L. is the corresponding author. E-mail: leon- 0.3048 ft m 3.2808 [email protected]. 0.0929 ft2 m2 10.7639 This is an open access article distributed under the CC 2.54 inch(es) cm 0.3937 BY-NC-ND license (https://creativecommons.org/ 25.4 inch(es) mm 0.0394 licenses/by-nc-nd/4.0/). 6.4516 inch2 cm2 0.1550 https://doi.org/10.21273/HORTTECH04324-19 1 micron(s) mm1
554 • October 2019 29(5) canarygrass (Phalaris arundinacea)is and, later, oryzalin were used to convert suspected plants identified using guard an invasive grass species on the Turn- several tropical landscape species to cell measurements is then verified by bull National Wildlife Refuge in the tetraploid forms by treating the meri- performing root tip chromosome state of Washington (Canwell 2015). stems of seedlings with saturated, small counts, a laborious and time-consum- The studies reported here deal cotton balls (Leonhardt and Shi, 2009). ing process. Today, more researchers with reducing or eliminating seed pro- Colchicine is an alkaloid and oryzalin is rely on flow cytometry (FCM) for the duction as a means of invasiveness, a dinitroaniline herbicide. Colchicine screening of treated plant material which is not meant to imply that clonal has proved to be a useful tool in many and ploidy-level confirmation. FCM propagationcannotalsoleadtoinva- breeding programs aimed at producing allows for the quantification of plant siveness. Kahili ginger (Hedychium superior cultivars (Kamemoto, 1985; nuclear DNA, subsequently provid- gardnerianum), yellow ginger (H. fla- Kamemoto et al., 1997; Tambong ing the user with the ploidy level of vescens), and white ginger (H. coronar- et al., 1998; Vainola, 2000), and pro- the samples screened. ium) are seed-producing, herbaceous, ducing sterile triploids by breeding FLOW CYTOMETRY. Atypicalflow perennial monocots but have massive induced tetraploids with diploids (Bla- cytometer contains several compo- spreading mats of short stout rhizomes kesley et al., 2002; St. Marseille and nents: a light source, a flow chamber that crowd out seedlings of native and Grant 1997). Similarly, oryzalin has and optical assembly, photodetec- exotic species in wet forest, roadsides, been shown to double the chromo- tors and processors to convert light open areas, and streamsides (New Zea- some numbers in catnip [Nepeta sp. signals into analog electrical im- land Plant Conservation Network, (Mitrofanova et al., 2003)], alocasia pulses, analog-to-digital convertors n.d.). This appears to be the primary [Alocasia sp. (Thao et al., 2003)], (ADCs), and a computer system for means of their invasiveness in Hawaii. rhododendron [Rhododendron hy- the analysis and storage of digitized Water lily (Nymphaea sp.) and hybrids brids (Vainola, 2000)], and lily [Lil- data. Essentially, the plant nuclei are may produce seed, but their coloniza- ium sp. (van Tuyl et al., 1990)]. extracted from young leaf tissue us- tion of ponds, ditches, canals, and slow- Research has shown that colchicine ing an extraction buffer and labeled moving streams in Hawaii is mostly and oryzalin have a similar mode of with a fluorescent dye (Dirihan et al., caused by their rapid spreading rhi- action at the molecular level. Both 2013; Oates et al., 2014). The sam- zomes and sometimes stolons, and in agents bind to plant microtubules ple is then loaded into the cytometer some tropical day-blooming varieties, that are involved in chromosome and illuminated, causing the dye to the viviparous production of clonal migration. Both agents disrupt mi- absorb the illuminating light and plantlets at the connection of petiole tosis by inhibiting spindle fiber for- fluoresce. The emitted light is then and leaf pad (personal observations). mation at metaphase (Strachan and converted to electric current pulses, The common coral tree (Erythrina Hess, 1983), resulting in an increase whicharefedtoamplifiers,digitized ·sykesii) is an invasive species in parts in the chromosome number of the using the ADCs, and stored in the of Australia, although it is a nonseed- daughter cells. The new growth from computer in the form of a histogram. producing sterile hybrid (putatively treated meristems is compared with CyStain PI Absolute P DNA E. coralloides · E. lysistemon). Logs, the new growth of untreated con- Staining Kit for Plant Genome Size branches, twigs, and other vegetative trols for morphological differences. (Partec, Munster, Germany) was used parts spread during flash floods and If there are discernable differences, for nuclei extraction and DNA stain- propagate into new plants. Distribu- those plants are subjected to guard ing of nuclear DNA from young leaves tion has also been aided by the cell screening (Leonhardt and Shi, used for flow cytometric measure- dumping of garden waste (Queens- 2009). ment. About 1 cm2 of young leaf tissue land Government, 2016). GUARD CELL MEASUREMENTS. was chopped for 30 to 60 s in a 500- The objective was to produce tet- Measuring stomatal guard cells re- mL ice-cold nuclei extraction buffer raploid forms of three species of shower quires producing an imprint of the with a sharp doubled-edge razor blade trees [pink shower tree (Cassia bakeri- abaxial side of the leaf of interest. The in a 55-mm plastic petri dish. The ana), golden shower tree (C. fistula), imprint is then viewed under the mi- slurry was then filtered through a 50- and white shower tree (C. javanica)], croscope and the size of the guard cells mm filter (Cell Trics, Partec) and the royal poinciana tree, african tulip tree, is measured. The theory behind this suspension of released nuclei was monkey pod tree, and indian coral tree method of screening for changes in stained in a solution composed of (Erythrina variegata), and cross them ploidy levels is simple. The cell volume staining buffer, propidium iodide, to diploid forms of the same species to of a plant is directly proportional to the and RNAse for a final volume of 2 mL. produce sterile triploid plants that amount of DNA present in the cell, so The relative fluorescence of to- would be low maintenance for lack of that doubling the amount of DNA, tal DNA of single nuclei was ana- seed pod litter and would not become which occurs when diploids are con- lyzed using a flow cytometer (Altra; invasive. The hypothesis was that the verted to tetraploids, causes the cell to Beckman-Coulter, Miami, FL), using tetraploid state could be achieved with double its volume. Doubling of the the 488-nm line of an argon ion laser the use of the mitotic inhibitors colchi- volume allows an increase in the size (I90C; Coherent, Santa Clara, CA) set cine and oryzalin. of the cell in any one dimension by at 200 mW. Control diploid plants 1.25, and comparisons of guard cells were used as external standards, and Materials and methods among a batch of chemically treated these standards were run intercalated In a series of experiments con- plants allows for the identification of between samples. The linear, log, and ducted by the author beginning in possible polyploids (Russell, 2004). peak fluorescence signals, along with 2005, the chemical mutagens colchicine Typically, the ploidy level of the forward- and side-scatter signals of the
• October 2019 29(5) 555 WORKSHOP propidium iodide-stained nuclei were In another plot at the UHW, 10 MONKEY POD TREE. Fifteen au- collected using a 610-bandpass filter, tetraploids, 5 diploid controls, and 19 totetraploids, three diploids, and five allowing 610 nm light from propi- plants thought to be mixoploids with plants thought to be mixoploids were dium iodide-bound DNA to enter both 2N and 4N tissues of the re- randomly planted 30 ft on center in the fluorescence detector. cessive yellow form of the african tulip a grove at the UHW in 2007 and SHOWER TREE SPECIES. At the tree were planted in a randomized began flowering within 5 years. Al- University of Hawaii Experiment Sta- block design and are now flowering. though the diploid plants of this tion at Waimanalo (UHW), Oahu, The diploid species is self-incompati- cross-pollinated species produced four golden shower trees (Fig. 1), ble. Seed will be harvested and sown pods 20 cm long, with 15 to 20 four pink shower trees, and six pink and seedlings will be run through the seeds, some of the tetraploid trees and white shower trees are all com- FCM protocol. Triploid seedlings will produced shriveled and seedless fruit pletely sterile as autotetraploids. They be grown to maturity and assessed for (Fig. 2) or fruit as small this grove and have been flowering profusely for several sterility. 200 seedlings were produced. FCM years, but none of them have produced any fruit. Diploid control plants of each species in the same plot are prolific producers of seed pods. Shower tree pod litter must be raked up from lawns before mowing. The littered pods are a nuisance because they are unsightly and often contain a foul-smelling resin (the pink shower tree is particularly malodorous). Like any plant with high seed production, they also have the potential to become invasive. This prob- lem might be avoided entirely by using the autotetraploid forms. These cultivars have no ability to become invasive, making them potentially desirable land- scape ornamentals if they could be readily propagated. Unfortunately, only Fig. 1. Golden shower tree. Seed-laden diploid control (left) and completely sterile four of 200 air layers on the autotet- autotetraploid (right). Unfortunately, the sterile form is not easily propagated. raploid forms rooted, and more than 50 grafts of tetraploid scions onto diploid rootstocks of the same species have failed. Clearly, more research into clonal propagation of polyploid shower trees is needed. ROYAL POINCIANA TREE. One royal poinciana tree was successfully converted to the tetraploid form in 2006 and has been flowering at the UHW for at least 6 years. It covers an area of 800 ft2. Diploid trees of similar size produce about 900 to 1000 seed pods/year. The 3-year average pod pro- duction for the tetraploid tree is 60 pods, with 70% seed abortion. No attempts have been made yet to clone this tree. AFRICAN TULIP TREE. One orange form of the african tulip tree was successfully converted to a tetraploid form. This 12-year-old tree has pro- duced fewer than 15 fruit since it began flowering 7 years ago, whereas diploid forms of comparable size and age pro- duce many hundreds of fruit annually. Reduced or complete cessation of seed production was especially important for the african tulip tree because it is listed as a highly invasive species (Staples et al., Fig. 2. Monkey pod seed pods from a diploid control tree (left) and from 2001). Clonal propagation has not yet a converted tetraploid tree (right). These pods are seedless. The tree, however, been attempted. does not consistently produce seedless pods.
556 • October 2019 29(5) results on 158 seedlings identified 11 triploid plants (6.96%) that are now 2 m tall and ready for field planting. These plants are expected to be sterile and without seed pods. Attempts to clonally propagate polyploidy mon- key pod trees have not yet been done. Results A reduction in seed fecundity of 95% to 100% was achieved in several converted species (Leonhardt, 2016, 2017b; Leonhardt and Shi, 2009). These results align with other reports of manipulated ploidy to reduce seed production of invasive species (Oates et al., 2014; Ranney, 2000). The use of polyploidy in breeding programs may be a useful strategy to reduce the harm caused by invasive species. P OLYPLOIDY AS AN INSECT PEST MANAGEMENT STRATEGY. While attempting to create a sterile form of the once popular indian coral tree, it was discovered that polyploid forms of this species are tolerant to an in- vasive insect pest that has nearly erad- icated the diploid species from the Hawaii landscape. The erythrina gall wasp (EGW; Quadrastichus erythrinae) was first collected in Hawaii in 2005 on the indian coral tree, a then-popular land- scape species. It quickly spread to all islands, causing severe damage to the native wiliwili (Erythrina sandwicen- sis) as well as to most introduced Fig. 3. Erythrina gall wasp (EGW)-susceptible indian coral tree (top left) and species. Galls on leaves, petioles, and EGW-tolerant mixoploid (top right). The mixoploid plant has 23.2% tetraploid stems are induced by the larvae of the nuclei and 9.6% octoploid nuclei, as determined by flow cytometry. It was tiny EGW. Leaves curl and become introduced to the landscape trade with the cultivar name Adrien. The histogram massively deformed, and petioles and (bottom) illustrates the relative quantities of diploid and polyploid nuclei in stems become swollen. Heavy infes- ‘Adrien’. tations caused defoliation and death of trees (Heu et al., 2008). Many thousands of trees have been killed by this pest. Polyploid individuals of the indian coral tree showed a high level of tolerance to the EGW, whereas in the same field plot, diploid individ- uals were destroyed. The induction of polyploidy in the EGW-susceptible indian coral tree has produced the EGW-tolerant cultivar Adrien (Fig. 3), which was introduced to the Hawaii landscape industry in 2017 (Leonhardt, 2017a). It is read- ily propagated by cuttings. Discussion The woody tree species discussed here were amenable to ploidy manip- ulation and induced sterility. Some of Fig. 4. Flowers from the royal poinciana tree. (Left) Diploid control. (Right) the polyploid plants are now more than Converted tetraploid.
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12 years old and show few differences invasive populations of Lythrum salicaria at 1 Feb. 2017.
558 • October 2019 29(5)