The Journal of Cotton Science 11:60–67 (2007) 60 http://journal.cotton.org, © The Cotton Foundation 2007 MOLECULAR BIOLOGY AND PHYSIOLOGY Towards Improved Cell Cycle Synchronization and Chromosome Preparation Methods in Cotton Randal A. Halfmann, David M. Stelly*, and David H. Young ABSTRACT biotechnological means are being sought throughout the world. Efforts to characterize the cotton genome Cotton is an economically important crop that are expanding domestically and internationally needs more extensive genetic characterization. (International Cotton Genome Initiative, 2004) and Highly effective methods for consistent cell cycle prospectively include complete genome sequencing. manipulation are needed to efficiently produce Chromosome manipulation and cytogenetic analysis high quality cytological preparations of chromo- are essential to germplasm introgression, genetic somes, and to improve methods of chromosome analysis, and genomics, all of which are vital to doubling, both of which impact cotton genomics breeding and breeding-related research. Procedures and breeding. This manuscript reports a procedure that enable reliable production of high quality mitotic for cell cycle synchronization of root tips using hy- chromosome preparations from cotton will help render droxyurea (HU), and the efficacy of several known the species more amenable to diverse protocols, e.g. chemicals for metaphase accumulation. Experi- cytogenetic analysis, chromosome doubling for ploidy mental treatments were administered hydroponi- manipulation, and harvesting of chromosomes for cally, followed by cytological determination of the flow sorting and chromosome-specific BAC library metaphase index. An 18-hour treatment of HU development. Development of these capabilities in at 3.5 mM gave a maximum synchronization of cotton would be facilitated by the development of about 6%. Seedlings were treated with five antitu- suitable cell synchronization procedures. bulin compounds of diverse chemistry; colchicine, A variety of methods have been developed to in- amiprophos-methyl (APM), nitrous oxide gas, a duce cell cycle synchrony in plant systems. Nutrient benzamide designated RH-4032, and a novel phe- starvation followed by transfer to fresh medium has nylcyclohexene colchicine mimic RH-9472. Three been used with some success in suspension-cultured of the antitubulin compounds, amiprophos-methyl, cells (Arumuganathan et al., 1991). Synchrony is RH-9472, and RH-4032, provided average meta- critically dependent on the state of the cells, so most phase indices of 0.3 or higher on synchronized root methods employ DNA synthesis inhibitors that have tips and were preferable to colchicine, the standard nominal affects on cell viability. One of the most agent for metaphase accumulation. The chromo- commonly used inhibitors is hydroxyurea (HU), some dispersing effects of nitrous oxide allowed the which targets the nucleotide biosynthesis pathway production of high quality chromosome prepara- needed for DNA synthesis by reversibly inhibiting tions, although nitrous oxide showed little ability ribonucleotide reductase and the production of de- to increase metaphase indices or to act concertedly oxyribonucleotides and ribonucleotides. Treatment with the other mitotic blocking compounds, which periods are generally longer than the length of the cell tend to cause chromosome aggregation. cycle, and result in the accumulation of cycling cells that have been arrested in late G1 to early S phase. otton (Gossypium hirsutum L.) is an economically Removal of the inhibitor allows the arrested cells to Cimportant fiber and oilseed crop. Because of its resume cycling in a synchronous manner. value, genetic improvements through conventional and Following synchronization, cells can be accumu- lated in metaphase by a treatment that is antagonistic to chromosome movement. Disruption of the spindle R. A. Halfmann and D. M. Stelly, Department of Soil and is a common approach, because it eliminates ten- Crop Sciences, Texas A&M University, College Station, sion on centromeres and invokes a tension-reporting TX 77843; D. H. Young, Dow AgroSciences LLC, 9330 metaphase/anaphase cell cycle checkpoint. Disruption Zionsville Road, Indianapolis, IN 46268 of the mitotic spindle apparatus typically involves *Corresponding author: [email protected] treatment with an antitubulin chemical and/or cold HAlFmANN eT Al.: Cell CyCle SyNCHRONizatiON AND CHROmOSOme PReP 61 temperature. The length of the treatment generally solubility in aqueous solutions than dinitroanilines correlates with the percentage of cells in arrested (morejohn and Fosket, 1991). Carbamate herbicides metaphase, although longer treatments are associated interact with plant tubulin in a poorly understood with chromosome decondensation and chromosomes manner, but the effect of these herbicides on plant splitting into chromatids (Dolezel et al., 1992). Cyto- microtubules is generally much less than APm and toxicity of the chemicals makes extended treatment the dinitroanilines (Morejohn and Fosket, 1991). undesirable. The most popular antitubulin chemical is APm was more effective than oryzalin, trifluralin, colchicine, despite being notoriously inconsistent for and pronamide for chromosome doubling in Beta plant cell cycle synchronization (Bordes et al., 1997). vulgaris L. ovule culture (Hansen et al., 1998). Colchicine is known to be more potent toward mam- Two novel chemicals, 3,5-dichloro-N-(3- malian cells than toward plant cells, which makes it chloro-1-ethyl-1-methyl-2-oxopropyl)-benzamide, a strong mammalian toxin and mutagen that must be designated RH-4032 (young et al., 2000), and trans- used with extreme caution. 4-nitro-5-(2,3,4-trimethoxyphenyl)cyclohexene, a Alternatives to colchicine have been developed colchicine mimic designated RH-9472 (young et that are largely specific to plant tubulin. Antimi- al., 2001), have been developed with significantly crotubule herbicides belong to a few chemically increased affinity for plant tubulin. in assays for diverse classes, including the dinitroanilines (e.g. inhibition of tobacco root growth, both RH-4032 oryzalin and trifluralin), the phosphorothioamidates and RH-9472 strongly inhibited elongation and pro- [e.g. amiprophos-methyl (APm)], the benzamides/ duced swollen root tips, a characteristic feature of carbamates [e.g. pronamide, chlorpropham, and antitubulin herbicides (Vaughn and lehnen, 1991), isopropyl N-(3-chlorophenyl) carbamate], as well and each chemical was more potent than all other her- as others (reviewed in Molin and Khan, 1997 and bicides that were compared (trifluralin, pronamide, Vaughn, 2000). zariliamide, APm, and chlorpropham for RH-4032, Concentrations necessary for significant effects and trifluralin and pronamide for RH-9472). Another in vivo are typically much lower than concentrations method for mitotic disruption is the use of nitrous needed to inhibit taxol-induced polymerization of pu- oxide, a nontoxic gas that in maize has proven more rified plant tubulin m( orejohn and Fosket, 1991). This effective than colchicine, trifluralin, APm, and tem- has been attributed to the lipophilic nature of these perature pretreatments for metaphase accumulation chemicals, which facilitates their uptake and allows for and chromosome counting (Kato, 1999). high effective concentrations at sites of microtubule Several reports have linked the use of microtu- nucleation, i.e. the nuclear envelope (Stoppin et al., bule disruptors with chromosome clumping. Chro- 1994) and cell cortex (Chan et al., 2003). The observa- mosomes treated at metaphase tend to aggregate tion that lipid levels correlate with tolerance to these tightly into clumps, making them impossible to chemicals, which may explain why dicots are less distinguish by microscopy or separate by flow cy- susceptible than monocots (molin and Khan, 1997), tometry (Lysak et al., 1999). Lee et al. (1996) noted is perplexing. Particularly relevant to applications in that chromosomes began to clump with trifluralin cotton is herbicide partitioning into lipid-rich gossypol treatments over five hours, and recommended that glands (Stegink and Vaughn, 1988). treatments be kept as short as possible to minimize Many reports have compared potencies of these the problem. lysak et al. (1999) and Dolezel et al. agents and their relative utility for botanical stud- (1999) reported that overnight incubation of APm- ies. Hess and Bayer (1974) specifically studied the treated roots in ice water improves chromosome effect of trifluralin on cotton microtubules. Some spreading for several cereal species. polyploidy and polymorphonucleate cells resulted In contrast, Kato (1999) indicated that nitrous from formation of restitution nuclei, but treatments oxide does not cause the chromosome-clumping were prone to yield an undesirable range of mitotic side-effect. in fact, it disperses maize metaphase disruption in single samples attributed to the ex- chromosomes so that the distances among chromo- ceedingly low water solubility of trifluralin. The somes increase with higher treatment pressures. This phosphorothioamidate herbicides are symptomati- makes nitrous oxide a potentially valuable pretreat- cally indistinguishable from the dinitroanilines and ment agent when used in combination with other bind to the same site(s) on α- and β-tubulin (Blume mitotic blocking compounds, if the dispersal effect et al., 2003) but have greater than 100-fold higher can overcome chromosome aggregation. JOURNAl OF COTTON SCieNCe, Volume 11, issue 1,