MISCELLANEOUS HORTSCIENCE 52(7):1029–1032. 2017. doi: 10.21273/HORTSCI12024-17 one or more spindle fiber inhibitors to allow the cell cycle to continue to metaphase while arresting cytokinesis. In addition, root tip Improved Method of Enzyme Digestion cold treatments have been used to help arrest cells at metaphase and condense chromo- for Root Tip Cytology somes (Jauhar, 2003). Next, the root tips are 1 1 2,3 fixed in a solution that arrest the cell cycle Jason D. Lattier , Hsuan Chen , and Ryan N. Contreras and are stored in an aqueous ethanol solu- Department of Horticulture, Oregon State University, 4017 Agriculture and tion until observation. For the root squash Life Sciences Building, Corvallis, OR 97331-7304 step, the roots are hydrolyzed in hydrochloric acid or a combination of hydrochloric acid Additional index words. chromosome counts, root squash, ploidy, ultraviolet resin, modified and ethanol. Alternatively, cell walls may be carbol fuchsin stain broken down by enzyme digestion using Abstract. Chromosome numbers are an important botanical character for multiple fields combinations of cellulase, cytohelicase, and of plant sciences, from plant breeding and genetics to systematics and taxonomy. pectolyase. Chromosome stains vary incl- Accurate chromosome counts in root tips of woody plants are often limited by their uding modified carbol fuchsin, Feulgen, small, friable roots with numerous, small chromosomes. Current hydrolysis and enzyme Giemsa, and acetocarmine, as well as fluo- digestion techniques require handling of roots before the root squash. However, optimum rochrome stains (Bationo-Kando et al., chromosome spread occurs when the cell walls have degraded past the point of easy 2016; Contreras et al., 2010; Jones et al., handling. Here, we present a new enzyme digestion protocol that is fast, efficient, and 2007; Rothleutner et al., 2016; Schneider flexible. This protocol reduces handling of the roots allowing for long-duration enzyme et al., 2015). digestion. Digestions are performed on a microscope slide, eliminating the need for In our observations, to obtain the highest handling digested cells with forceps or pipettes. To illustrate the flexibility of this method quality chromosome spread, tissue degrada- across woody plant taxa, we performed chromosome counts on five angiosperms and one tion often must proceed beyond the point gymnosperm. Ploidy levels included diploids, triploids, and tetraploids with chromosome which the root tip can be easily handled and numbers ranging from 2n =16to2n = 80. The range of holoploid 2C genome sizes still remain intact. However, most protocols spanned 1.54–24.71 pg. This protocol will provide a useful technique for plant cytologists using enzyme digestion require handling working with taxa that exhibit a wide range of genome size and ploidy levels. after hydrolysis or digestion. This creates a problem that can be solved by digesting the excised root tip on the same surface used Genome size, chromosome number, and and tropical woody species (Bationo-Kando to perform the root squash. Once the cell ploidy level are important biological param- et al., 2016; Cai et al., 2013; Dahmer et al., walls have been fully digested, the weight of eters for plant breeding, systematics, and 2009; Schneider et al., 2015). Traditional the cover slip on to the root tip should be evolution. Since the first published chromo- cytology can be used for identifying aneuploids sufficient to initially spread the cells. In the some counts of plants in 1882 (Garbari et al., (additional or missing chromosomes) (Hu et al., current study, we report a novel root squash 2012), 25% of angiosperms have been 2015) or dysploids (alterations from chromo- protocol from the Ornamental Plant Breeding measured for chromosome number (Castiglione some fusion or fission) difficult to detect using Laboratory at Oregon State University that and Cremonini, 2012), and 2.1% have flow cytometry (Rockinger et al., 2016). To has proven to be a fast, effective, and adjust- measurements of genome size (Garcia et al., meet the demand for accurate chromosome able root tip cytology method applicable across a wide range of woody plant taxa. 2014). Holoploid 2C genome sizes of plants counts, traditional cytology has proven useful span about a 2400-fold range, from 0.13 pg in the development of databases across taxo- (Genlisea margaretae Hutch.) to 304.46 pg nomic groups and formation of data sets for Materials and Methods (Paris japonica Franch.) (Bennett and meta-analysis of chromosome number across Plant material. Six taxa were investigated Leitch, 2011; Chen et al., 2014; Fleischmann plants and animals (Peruzzi et al., 2014). to represent a wide range of genome size et al., 2014; Pellicer et al., 2010). Although traditional cytology has been an and chromosome number, including five an- Traditional cytology has many uses in invaluable tool for studies in plant genetics, giosperms and one gymnosperm (Table 1). modern studies on woody plants. Combined cytology on woody plants can be challeng- with flow cytometry, cytology has been used Plants were grown in a temperature-controlled ing compared with their herbaceous coun- glasshouse at Oregon State University. Al- to calibrate genome size with ploidy level terparts. In general, many woody plants and to confirm chromosome and ploidy though variable growth conditions would possess small, friable roots with numerous likely affect the quality of roots for cytology, variation among related taxa and hybrids in small chromosomes making cytology par- both temperate woody species (Contreras et al., all plants were grown under the same stan- ticularly difficult (Lattier et al., 2013). dard glasshouse conditions. Plants were 2007, 2009, 2010; Gillooly and Ranney, 2015; Chromosome counting techniques can be Jones et al., 2007; Lattier et al., 2013; Oates container-grown in a 2:1 mixture of Metro- tedious and require experienced histologists Mix Professional Growing Mix (Sun Gro et al., 2014; Parris et al., 2010; Ranney et al., (Ochatt, 2008). In the modern era, cytoge- 2007; Rothleutner et al., 2016; Rounsaville and Horticulture, Agawam, MA) and Perlite (Su- netic studies in hardwood trees have not kept Ranney, 2010; Shearer and Ranney, 2013) preme Perlite Company, Portland, OR). pace with current genomic studies because Plants were initially hand-watered using mu- of small genomes and relatively small chro- nicipal water on an as-needed basis and mosomes (Ribeiro et al., 2008). Traditional substrate solution pH and electrical conduc- Received for publication 12 Apr. 2017. Accepted cytology is also necessary for chromosomal tivity (EC) were routinely monitored using for publication 27 May 2017. fluorescent labeling techniques, such as the pour-through nutrient extraction proce- We gratefully acknowledge our sources of plant fluorescent in situ hybridization and geno- dure (Wright, 1986). Once the substrate so- material: the US National Arboretum, Rogow mic in situ hybridization, and has proven lution EC was less than EC = 1.0 mS·cm–1, the Arboretum, NCGR-Corvallis, Monrovia Nursery, valuable for characterizing hybrids in plants were fertigated at each irrigation with and Blue Heron Farm. Additional technical support woody plants with small chromosomes Peters Professional 20N–4.4P–16.6K plus provided by Tyler Hoskins and Sawyer Hurd in (Van Laere et al., 2010). production and maintenance of the plant materials. micronutrients (Everris NA Inc., Dublin, 1Graduate Research Assistant. Current root tip cytology consists of three OH), calibrated such that irrigation water –1 2Associate Professor. broad steps (prefixative, fixative, and root EC = 1.0 mS·cm . Plants were grown in 3Corresponding author. E-mail: ryan.contreras@ squash) with slight variations for each step. a glasshouse with set temperatures of 24 °C oregonstate.edu. For the prefixative step, roots are treated with day/17 °C night with a 14-h photoperiod. HORTSCIENCE VOL. 52(7) JULY 2017 1029 Reported chromosome numbers on the occasionally. This step was repeated twice hydration. To localize enzyme digestion to taxa investigated span a range of 2n =16to for a triple rinse of each root over a total of the microscope slide, a circular well was 2n = 80 and holoploid genome sizes of 1.54– 15 min. Each root was placed on a clean slide created using an ultraviolet resin pen (BondicÒ; 24.7 pg. Ribes sanguineum Pursh is reported under a dissecting microscope (SMZ1500; Niagra Falls, NY). A circle of ultraviolet to be a diploid 2n =2x = 16 (Darlington and Nikon, Tokyo, Japan), and the root tip was resin was drawn around the root tip and Wylie, 1956) with a holoploid genome size excised (Fig. 1A). The remaining root was water droplet (Fig. 1B). To facilitate removal of 1.94 pg (OPBL, unpublished data). Roots discarded, and excess water was removed of the circular well, a ‘‘pull tab’’ of the were collected from an open-pollinated seed- with a single-ply, low-lint tissue (VWR In- ultraviolet resin was placed over a small ling of R. sanguineum ‘Pokey’s Pink’. Quer- ternational, Radnor, PA) leaving only a small piece of wax paper (Fig. 1B). The slide was cus robur L. is reported to be a diploid 2n =2x = droplet encompassing the root tip to maintain then placed in an ultraviolet crosslinker (CL 24 with a holoploid genome size of 1.85 pg (Favre and Brown, 1996). Roots were col- lected from an open-pollinated seedling of Table 1. Digestion times for taxa investigated using an improved enzyme digestion protocol for root tip the columnar Q. robur ‘Fastigiata’. Thuja cytology. occidentalis L. is reported to be a diploid 2n = 2x = 22 with a holoploid genome size of Digestion 2C genome Taxaz time (h)y 2C formulax size (pg)w 24.71 pg (Hizume et al., 2001). Roots were Cercidphyllum japonicum ‘Rotfuchs’ Red Fox 0.5 2n =2x = 38 1.53 sampled from an open-pollinated seedling Ribes sanguineum ‘Pokey’s Pink’ 0.5 2n =2x = 16 1.94 collected from a plant growing at the Lewis Acer tataricum subsp.