Oryzalin-induced Tetraploidy in Cryptomeria japonica (Cupressaceae)

Japanese-cedar [Cryptomeria japonica (L.f.) D. Don] represents an alternative to leyland cypress [·Cuprocyparis leylandii (A.B. Jacks. & Dallim.) Farjon] as an evergreen screen or specimen plant for landscapes. It performs well under a range of soil and environmental conditions but has been underused attributable, in part, to unsightly winter browning caused by photoinhibition. In previous studies, chance seedlings that did not exhibit winter browning were identified as tetraploids. The current study was conducted to induce polyploidy in japanese-cedar. Approximately 600 seedlings were sprayed with 150 mM oryzalin + 0.1% SilEnergy for 30 consecutive days under laboratory conditions. Two hundred thirty-seven seedlings with thickened and twisted leaves were selected, transplanted, and grown in a glasshouse for 120 days. Seedling ploidy levels were analyzed using flow cytometry 180 days after treatment (DAT), identifying 197 (83.1%) tetraploids, 22 (9.3%) cytochimeras, and 18 (7.6%) diploids. Morphology of induced tetraploids was similar to that previously described and provided a phenotypic marker during selection that was over 92% accurate. A random subset of 20 tetraploid individuals was analyzed 270 DAT and were found to contain only tetraploid cells in the leaves analyzed, confirming stability over this period. This study demonstrated the use of oryzalin for inducing tetraploids in japanese-cedar, which we predict will be effective in other gymnosperms. Cryptomeria D. Don is a monotypic genus comprised of C. japonica and its two varieties, C. japonica var. japonica and C. japonica var. sinensis Miq. Cryptomeria is native to China (var. sinensis) and Japan (var. japonica) and is an important timber tree of the latter, reaching heights of 36 to 46 m in the wild (Dallimore and Jackson, 1967) but is typically 15 to 25 m in gardens (Tripp and Raulston, 1992). Cryptomerias, also commonly called japanese-cedars or sugi, offer an alternative to leyland cypress due to their limited pest problems (Tripp, 1993; Tripp and Raulston, 1992), ability to perform well under the hot, humid conditions of the southeastern United States, and tolerance of heavy soils during both wet and dry conditions (Tripp, 1993). Cultivars are numerous and varied and include forms that are dwarf, have irregular branching, and have varying foliage colors, including variegated (Rouse et al., 2000). However, in full sun, they exhibit browning in winter that can be undesirable (personal observation). Winter browning in japanesecedar occurs in sun-exposed leaves during periods of low temperature indicating that photoinhibition plays a role (Han and Mukai, 1999; Ida, 1981). Ida (1981) reported that chloroplasts in sun-exposed leaves were transformed into rhodoxanthin-containing chromoplasts during winter, resulting in the brown–red color. In Japanese forestry nurseries, tetraploid forms of japanese-cedar have been identified based on their morphology (Chiba, 1951). Tetraploids have been shown to be more resistant to oxidative damage from ultraviolet light than diploids through increased antioxidant levels (Niwa and Sasaki, 2003). However, these seedlings have been rogued from nurseries based on their unfamiliar appearance (Chiba, 1951) and saplings that readily change needle color from green to brown or orange–yellow have been recommended as being superior individuals for silviculture (Han and Mukai, 1999). Negative selection has been imposed based on the lack of use of tetraploids in a silviculture, but they may have great potential for use in the nursery and landscape industries in the United States by providing nonbrowning forms that consumers find more attractive. Polyploidy in gymnosperms is rare (Ahuja, 2005; Khoshoo, 1959, 1961), but following the work of Blakeslee and Avery (1937), there were a number of studies that attempted to induce polyploidy. The primarily goal of those studies was to develop more vigorous trees for timber production. Research included attempts to induce polyploidy in pines (Pinus L.) (Hyun, 1953; Mergen, 1959; Mirov and Stockwell, 1939), japanese cypress (Chamaecyparis obtusa Endl.) (Kanezawa, 1951), and giant sequoia [Sequoiadendron giganteum (Lindl.) J. Bucholz] (Jensen and Levan, 1941). These studies used a variety of delivery methods at various stages of development, including treatment of seeds, newly germinated seedlings, and shoot tips; however, all previous studies to induce polyploidy have used colchicine. Colchicine acts by disrupting the microtubular structure of dividing cells in both plants and animals (Bartels and Hilton, 1973). The mode of action of dinitroaniline herbicides such as oryzalin has also been determined (Bartels and Hilton, 1973; Upadhyaya and Noodén, 1977, 1980) and have been found to bind more specifically to plant tubulin in vitro than colchicine (Yemets and Blume, 2008). In addition to more specific binding, dinitroaniline herbicides have been found to be less toxic and to act at 100 (Yemets and Blume, 2008) to 1000 times (Upadhyaya and Noodén, 1977) lower concentrations than colchicine. As a result of the reduced toxicity and more effective binding, dinitroaniline herbicides such as oryzalin may be preferable for inducing polyploidy. The objective of the current study was to induce polyploidy in Cryptomeria japonica using oryzalin. Materials and Methods Plant material. Wild-collected seed from China of Cryptomeria japonica were received from Lawyer Nursery, Inc., Plains, MT. Based on the collection site, the seed used may have been C. japonica var. sinensis; however, this was not able to be confirmed. Approximately 1000 seeds were sown in germination trays containing 8 pine bark:1 sand (v/v) amended with 0.91 kg m dolomitic lime and 0.45 kg m Micromax (The Scotts Co., Marysville, OH) and allowed to germinate in a glasshouse with day/ night set temperatures of 27/20 C. Approximately 600 seedlings germinated and were moved to the laboratory and grown under constant light (28 mmol m s) supplied by cool-white fluorescent lamps at 25 C. Cytology. Roots were harvested and pretreated for 24 h in 3 mM 8-hydroxyquinoline (Fisher Scientific, Suwanee, GA) + 0.24 mM cycloheximide (Acros Organics, Morris Plains, NJ) at 4 C. After pretreatment, roots were transferred to Carnoy’s solution [6 100% ethyl alcohol:3 chloroform:1 glacial acetic acid (by volume)] and fixed overnight at 25 C. Roots were rinsed with deionized water and transferred to aqueous 70% ethyl Received for publication 1 Oct. 2009. Accepted for publication 25 Nov. 2009. We thank Amanda Webb, Nancy Hand, Bruce Tucker, and Brandon Coker for their technical assistance. From a dissertation submitted by R.N.C. as partial fulfillment of the requirements for the Ph.D. degree. Graduate Research Assistant. Current appointment and address: Assistant Professor, Department of Horticulture, Oregon State University, Corvallis, OR 97331-7304 Professor. Assistant Professor. To whom reprint requests should be addressed; e-mail [email protected]. 316 HORTSCIENCE VOL. 45(2) FEBRUARY 2010 alcohol (v/v) and stored at 4 C until observation. At the time of observation, roots were rinsed in deionized water for 30 min and root tips were excised on a glass slide using a razor. Root tips were then incubated at 37 C in an enzyme mixture [0.5% cellulase (Karlan Research, Torrance, CA), 0.5% cytohelicase (Sigma-Aldrich Co., St. Louis, MO), and 0.5% pectolyase Y-23 (Karlan Research), in 50 mM citrate buffer at pH 4.5] for 4.5 h. After digestion, root tips were rinsed, transferred to a glass slide using a Pasteur pipette, and liquid removed by wicking with a one-ply tissue. Meristematic cells were separated from root cap and other nondividing cells by gently pressing the root tip with a dissecting probe. Before allowing cells to dry, a drop of modified carbol fuchsin stain (Kao, 1975) was added and cells were spread under a cover glass by applying even pressure. Slides were scanned at ·100 magnification; counting and photomicrography were performed at ·1000 magnification using a compound light microscope (Fisher Micromaster I; Fisher Scientific). Chromosomes of five cells were counted. Inducing polyploidy. Seedlings were sprayed to runoff daily for 30 d with an aqueous solution containing 150 mM oryzalin (supplied as Surflan AS; United Phosphorus, Trenton, NJ) + 0.1% SilEnergy (Brewer International, Vero Beach, FL), an organosilicate surfactant, using a standard spray bottle (Model P-32; Sprayco, Farmington Hills, MI). The rate of oryzalin was selected based on several previous studies in other genera that induced polyploidy using 150 mM solutions (Contreras et al., 2007, 2009; Olsen et al., 2006). After 30 d, 500 surviving seedlings were moved to a glasshouse and transplanted into six trays containing the pine bark substrate described. Seedlings were allowed to grow for 4 months and then transplanted into 0.2-L containers filled with 1 Pro-Mix BX with Biofungicide (Premier Horticulture, Quakertown, PA):1 pine bark/ sand mixture described previously (v/v), and fertilized weekly at a rate of 100 ppm nitrogen using TotalGro 20N–4.4P–17.6K watersoluble fertilizer (SDT Industries, Winnsboro, LA). Morphology indicative of polyploidy in japanese-cedar (Chiba, 1951) was used to select 237 seedlings for transplantation. After 1 month of growth [180 d after treatment (DAT)], the ploidy level of the seedlings was determined using flow cytometry. Identification of polyploidy. Flow cytometry, as described in Contreras et al. (2009) with modifications, was conducted on the 237 selected seedlings with altered leaf morphology. Five plants were evaluated during each analysis by bulking leaves. For bulked samples, a comparable amount of mature leaf tissue from five plants was collected and analyzed. In bulked samples in which a single peak was observed, all plants were recorded as being composed solely of tissue at the ploidy level corresponding to the fluorescence channel. For bulked analyses in which there were multiple peaks, plants were analyzed individually to determine ploidy of individual plants. The channel corresponding to diploid mean relative DNA fluorescence (MRF) was determined based on an individual that was confirmed as diploid (2n = 2x = 22) using cytology as described previously. The diploid channel served as a standard for comparison with determined ploidy for most samples. In some cases, the peak was ambiguous as a result of peak shifting and ploidy level was not clear. These samples were analyzed individually using an internal standard [(Pisum sativum L. ‘Ctirad’; 2C = 8.76 pg) (Greilhuber et al., 2007)]. The internal standard was used to calculate the 1C DNA content of the diploid [1C DNA (pg) = (MRF sample/MRF standard) · 8.76 pg)/2)]. DNA content of the unknown samples was calculated and ploidy determined based on the 1C DNA content of the diploid. A random subset of 20 individuals identified as tetraploid 180 DAT was reanalyzed using flow cytometry 270 DAT to determine if they remained stable tetraploids. Results and Discussion Results from chromosome counts on Cryptomeria japonica revealed a diploid complement of 2n = 2x = 22 (Fig. 1). This finding agrees with earlier results (Khoshoo, 1961; Sax and Sax, 1933). As a result of the large size and difficulty in spreading chromosomes, it was necessary to use the enzyme mixture described for digestion as opposed to a more standard HCl hydrolysis. Treating C. japonica seedlings for 30 consecutive days with 150 mM oryzalin + 0.1% SilEnergy successfully induced tetraploidy. Numerous preliminary experiments were unsuccessful in developing tetraploids, including soaking seeds in various rates of colchicine, germinating seeds in various rates of colchicine, and treatment of shoot tips of seedlings and stem cuttings (Contreras, 2009; Contreras and Ruter, 2008) with various rates of oryzalin. We hypothesized that previous applications of oryzalin were unsuccessful because they were too short (1 to 5 d) and did not contain a surfactant to penetrate the cuticle. A total of 237 seedlings was analyzed using flow cytometry and of these, 219 (92.4%) contained tetraploid cells (Table 1). Flow cytometric analysis of seedlings revealed 197 (83.1%) tetraploids, 22 (9.3%) cytochimeras, and 18 (7.6%) diploids (Table 1; Fig. 2). Morphology of induced tetraploids (Fig. 3), primarily thickened and twisted leaves, was similar to that previously described (Chiba, 1951) and provided a phenotypic marker for selection during transplantation that was over 92% accurate. Tetraploids had thicker leaves, but leaf length and growth rate displayed variation typical of a seedling population. Other populations grown from the same seed source had no individuals that exhibited leaf morphology indicative of polyploidy (personal observation), which provides strong indication that Fig. 1. Photomicrograph of a chromosome spread from a root tip squash of diploid (2n = 2x = 22) Cryptomeria japonica taken at ·1000 magnification. Scale bar = 10 mm. Table 1. Results of treating Cryptomeria japonica seedling shoot tips with 150 mM oryzalin + 0.1% SilEnergy for 30 consecutive days to