Improved Method of Enzyme Digestion for Root Tip Cytology

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 collected 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. ginnala 12n =3x = 39 2.34v Brown Horticulture Research Farm in Cor- Quercus robur ‘Fastigiata’ 2 2n =2x = 24 1.85 vallis, OR (field location 09.09). The original Hibiscus syriacus ‘Notwoodtwo’ White Chiffon 2 2n =4x = 80 4.70 seed source for this plant was accessioned as Thuja occidentalis 32n =2x = 22 24.71 10-0024 and was received from Lawyer zRoots were collected from open-pollinated seedlings from listed taxa. Nursery, Olympia, WA. Cercidiphyllum yOptimal digestion times for root tip cytology. x japonicum Siebold & Zucc. is reported to Ploidy and chromosome number of somatic root tip cells. wReported holoploid 2C genome size. be a diploid 2n =2x = 38 with a holoploid v genome size of 1.53 pg (Garcia et al., 2010). Estimated triploid 2C genome size based on published diploid and tetraploid genome sizes. Roots were collected from an open-pollinated seedling of C. japonicum ‘Rotfuchs’ (Red Fox) at the U.S. National Arboretum (Beltsville, MD). Acer tataricum subsp. ginnala (Maxim.) Wesm. is reported to be both a diploid 2n =2x =26(Darlingtonand Wylie, 1956) with a holoploid genome size of 1.65 pg (Lattier, 2016) and an induced autotetraploid 2n =4x = 52 with a holoploid genome size of 3.10 pg (Lattier, 2016). Roots were collected from an open-pollinated seedling (OP2016-04- 014) of a tetraploid cytotype (12-0011- 010) in an isolation block (field location 75.18) comprised a mixture of tetraploid and diploid cytotypes. Hibiscus syriacus L. is reported to be a tetraploid 2n =4x =80 (Darlington and Wylie, 1956) with a holoploid genome size of 4.70 pg (Contreras et al., 2013). Roots were collected from a self-pollinated seedling of H. syriacus ‘Notwoodtwo’ White Chiffon. Prefixative. Root tips were collected before 1000 HR after two consecutive sunny days and suspended in 1.5 mL microcentri- fuge tubes containing a solution of 2 mM 8-hydroxyquinoline and 0.24 mM cyclohexi- mide. The root tips were treated in the dark at room temperature for 2.5 h before a cold period in a refrigerator at 4 C for 2.5 h. Fixative. After 5 h in the prefixative solution, the root tips were transferred to a filter paper–lined glass funnel atop a Buchner€ (vacuum) flask. The root tips were thoroughly rinsed with filter-sterilized water Fig. 1. Steps in an improved enzyme digestion for root tip cytology. (A) Rinsed roots are placed on a clean and fixed overnight at room temperature in slide under a dissecting microscope and root tip is removed. (B) A ring of ultraviolet resin is placed Carnoy’s solution (6 parts 95% ethanol: 3 around the root tip and overlapping a waxed paper pull tab. The resin is set in an ultraviolet crosslinker, parts chloroform: 1 part glacial acetic acid; by and enzyme digestion solution is added to the well to encompass the root tip. (C) Enzyme digestion volume). After fixing the cells, the roots were takes place in a 37 C oven with the slide in a petri dish atop a small weighing dish and moist filter paper. (D) After digestion, the pull tab is used to remove the enzyme digestion well, and the root tip is transferred to a storage solution of 70% rinsed with a droplet of water before being wicked dry. (E) A drop of modified carbol fuchsin stain ethanol and stored in a refrigerator at 4 C. is added, and a glass cover slip is lowered on to the root tip with half of a double-sided razor blade. Root squash. The roots were transferred (F) The slide is covered with a bibulous paper, and the root is squashed using gentle pressure from from the storage solution to a small beaker a pencil eraser. (G) The prepared slide is stored at room temperature in the petri dish until viewed with of filter-sterilized water for 5 min, swirling a light microscope.

1030 HORTSCIENCE VOL. 52(7) JULY 2017 1000 ultraviolet Crosslinker; UVP, LLC, San Jose, CA). Bulk focus stacking of small Upland, CA) for 30 s to set the ultraviolet chromosomes can still leave individual chro- resin. Using a low-lint tissue, the droplet of mosomes out of focus. Therefore, fine editing water containing the root tip was wicked (selecting sharpest focus for individual chro- away, and a droplet of enzyme solution was mosomes) was performed when necessary pipetted on the root tip (Fig. 1B). The enzyme using GIMP 2.8.18 (GNU Image Manip- solution was composed of 0.5% cellulase ulation Program, https://www.gimp.org/). A (from Trichoderma reesei; Sigma, St. Louis, minimum of 15 highly resolved cells were MO), 0.5% cytohelicase (from Helix poma- observed per taxa. tia; Sigma), and 0.5% pectolyase (from Aspergillus japonicus; Sigma) in a sodium Results and Discussion citrate buffer at pH = 4.5. Next, the slide was placed in a humid Root squashes were successful across all environment to maintain the droplet through- six taxa. All roots were treated the same in the out the digestion period. Humidity was main- prefixative and fixative steps. However, we tained in a glass petri dish with a dampened recommend adjusting duration of prefixative filter paper; the slide was kept dry by resting treatment to each specific taxon, as recom- it on a small weighing dish (Fig. 1C). The mended in other woody plant cytology pro- glass petri dish containing the slide was tocols (Gamage and Schmidt, 2009). Because incubated at 37 C in an oven (IsotempÒ the duration of the mitotic cycle is positively Oven 655F; Thermo Fisher Scientific, Waltham, correlated with genome size, a longer prefix- MA). Multiple root tips per taxa were ative process should be used as genome sizes digested at varying durations until an optimal increase (Bennett, 1998; Schneider et al., digestion was achieved. Optimal digestion 2015). Duration of digestion was adjusted was achieved when the weight of the cover for each taxon (Table 1). We observed that slip provided enough force to break apart the the digestion time was influenced by the root root tip. Less than ten slide preparations per tip size and genome size of each taxa. taxa were required to find an optimal di- Optimal digestion times were taxon-specific gestion time for each taxon. and varied from 30 min for Ribes sanguineum Once digestion was complete, the glass to 3 h for Thuja occidentalis (Table 1). When petri dish and slide were removed from the calibrating the digestion step, under-digested oven. To prepare the root squash, the paper root tips failed to break apart under the ‘‘pull tab’’ was used to remove the ring of weight of the cover slip whereas over- ultraviolet resin (Fig. 1D). Excess enzyme digested root tips yielded cells with missing solution was wicked away before adding and far-spread chromosomes. Modified car- a drop or two of filter-sterilized water to the bol fuchsin proved a fast and effective stain root tip to rinse. The area surrounding the requiring no extra steps, such as heating the root tip was wiped clean with a low-lint tissue slide (required for acetocarmine), extended before wicking away excess water on the incubation (required for Fuelgen), or special Fig. 2. Photomicrographs from an improved en- surface of the root tip. A single drop of light sources (required for fluorochrome zyme digestion protocol for root tip cytology. modified carbol fuchsin stain (Kao, 1975) stains). Metaphase chromosomes observed at magnifi- was pipetted on the root tip (Fig. 1E). Using Chromosome counts of metaphase cells cation ·1000 from root apical meristems. m half of a double-sided razor blade, a bridge revealed diploid, triploid, and tetraploid cyto- Scalebar = 10 m. (A) Ribes sanguineum, was made to position a 22 · 22 mm cover slip types (Fig. 2). Ribes sanguineum was con- 2n =2x =16;(B) Quercus robur,2n =2x =24; (C) Thuja occidentalis,2n =2x =22;(D) Cerci- at an angle over the root tip and stain droplet firmed as a diploid 2n =2x = 16 (Fig. 2A). diphyllum japonicum,2n =2x =38(E) Acer (Fig. 1E). The cover slip was quickly lowered Quercus robur was confirmed as a diploid 2n tataricum subsp. ginnala,2n =3x =39; on to the slide to prevent bubbles and covered =2x = 24 (Fig. 2B). Thuja occidentalis was (F) Hibiscus syriacus,2n =4x =80. with a sheet of bibulous paper (Fig. 1F). confirmed as a diploid 2n =2x = 22 (Fig. 2C). Pressure was applied to the cover slip using Cercidiphyllum japonicum was confirmed as a pencil eraser while the bibulous paper a diploid 2n =2x = 38 (Fig. 2D). Acer interploidy crosses. Confirmation of a trip- wicked away excess stain (Fig. 1F). Slides tataricum subsp. ginnala was confirmed as loid cytotype in Acer tataricum subsp. gin- mounted with root squashes were placed a triploid (2n =3x = 39) resulting from an nala illustrates the utility of the protocol in back into petri dishes and stored at room interploid cross in our isolation block confirming hybrids from interploid crosses. temperature. This allowed for multiple slide (Fig. 2E). Hibiscus syriacus was confirmed Minimal handling of root tips combined preparations without sealing because the as a tetraploid 2n =4x = 80 (Fig. 2F). with long duration digestion makes this pro- humidity chambers prevented the slides from With chromosome estimates published tocol a practical method for root tip cytology drying (Fig. 1G). for only 25% of angiosperms (Castiglione in woody plants. Its simplicity makes it a fast Chromosome counts. All slides were and Cremonini, 2012), there is a clear need method for producing quality root squashes. screened for condensed chromosomes at for additional cytological studies. Chromo- Other hydrophobic barrier pens (PAP pens) a magnification of ·200 (·10 adapter and some counts are an important biological have been used for cytology. However, ul- ·20 objective) on a light microscope (Axio character necessary to multiple fields of plant traviolet resin pens are less expensive and imager.A1; Zeiss, Thornnwood, NY). Con- sciences, from plant breeding and genetics to leave little to no residue, unlike PAP pens densed and spread chromosomes were photo- systematics and taxonomy. Our protocol pro- which require xylene for residue removal. graphed under oil immersion at ·1000 (·10 vides an improved method for root tip cytol- Although we used a crosslinker, ultraviolet adapter and ·100 objective; AxioCam MRm, ogy that may contribute to the growing lamps and flashlights are ubiquitous and can Zeiss). To maximize resolution of each chro- number of chromosome surveys. The new be acquired for less than twenty dollars, mosome and extend depth of field, multiple enzyme digestion protocol proved an effec- making the price of this protocol compara- photos were taken per cell at different focal tive tool for producing high resolution meta- ble with previous methods. Its flexibility can distances. Bulk focus stacking was per- phase chromosomes across multiple woody be combined with other methods with formed using the Auto Blend feature in plant taxa. Accurate chromosome counting more complicated procedures, such as Photoshop CC 2015.5.1 (Adobe Systems, is critical when assessing wide hybrids or 4#,6-diamidino-2-phenylindole-staining, to

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