PLANT CYTOGENETICS

A citotaxonomia vegetal desenvolveu-se inicialmente nos países do primeiro mundo, acumulando dados e estabelecendo modelos evolutivos baseados principalmente em táxons e floras temperadas. A introdução de técnicas citogenéticas mais refinadas tem permitido uma análise mais acurada da variabilidade cariotípica, mas até o momento estes novos métodos têm sido aplicados apenas a um número muito pequeno de táxons. Assim, o principal instrumento do citotaxonomista continua sendo a variabilidade cromossômica numérica. A região tropical ainda é muito carente de dados, especialmente os trópicos americanos. No Brasil, a citotaxonomia se desenvolveu como parte da citogenética e não como uma abordagem taxonômica. Mesmo assim, nos últimos anos tem crescido o número de laboratórios trabalhando com citogenética vegetal e tem havido muitos avanços no estudo citotaxonômico de elementos de nossa flora.

Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes.

The recent realization that industrial effluents and wastes could pose health hazards to future generations has led to considerable research on the health effects of these substances and to efforts to regulate their release. The Research Conservation and Recovery Act (RCRA) specifically addresses the potential health and environmental hazards of solid wastes. Evaluation of long-term consequences of exposure to the huge number of potentially hazardous compounds and complex mixtures would tax the scientific community’s whole-animal testing capabilities. Therefore, many short-term bioassays have been developed to rapidly screen compounds for toxicity, mutagenicity, teratogenicity, and carcinogenicity. It is hoped that these short-term tests will predict lealth hazards early in the development of new technologies, allowing technological changes to be made at early stages, and resulting in timely whole-animal testing where screening indicates potential hazards. Time and money should thus be saved in both technological development and biological testing.

Epigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act in combination as a unique pattern. A total of 16 plant species belonging to 11 genera of eight families (five dicots and three monocots) including land plants, epiphytes (orchids) and the holokinetic taxa (Drosera spp.) were analysed for chromosomal distribution of dual modified antiphospho (Ser10)-acetyl (K14)-histone H3 (H3S10phK14ac) to understand the combinatorial chromatin dynamics during mitotic cell division in plants. The anti-H3S10phK14ac evidently mark the pericentromeric chromatin on mitotic chromosomes of the plants excluding the holokinetic Drosera species, which revealed the immunolabelling of whole chromosomes all along the arms. The dual modified immunosignals were absent during early stages of mitosis, appeared intensively at metaphase and remained visible until late-anaphase/telophase however, labelled the whole chromosomes during meiotic metaphase I. Colocalization of anti-H3S10phK14ac with an onion's CENH3 antibody on mitotic chromosomes of Allium revealed the chromosomal location of anti-H3S10phK14ac in the region between signals for CENH3 detection. Overall analysis suggests that the unique localization of combinatorial histone modification mark at pericentromeric chromatin might have attributed through 'phospho-acetyl' cross talk that ultimately facilitate the sister chromatid cohesion at pericentromeres following condensation events in mitotic chromosomes. Here, we propose that dual modified H3S10phK14ac histone may serve as an additional cytogenetic landmark to identify pericentromeric chromatin during mitosis in plants. The plausible role of histone cross talk and future perspectives of combinatorial histone modification marks in plant cytogenetics with special reference to chromatin dynamics have been discussed.

In situ hybridization is the direct hybridization of specific nucleic acid sequences to morphologically preserved cytological preparations such as inter-phase nuclei, metaphase chromosomes, cells or tissue sections. The detection of specific nucleic acids in situ with radiolabeled probes was first introduced by Gall and Pardue [ 1]. At this time, autoradiography was the only means of detecting hybridized sequences and because molecular cloning was not possible in those days in situ hybridization was restricted to those sequences that could be purified by conventional biochemical methods. In spite of the high sensitivity and wide applicability of in situ hybridization it was not until the development of molecular cloning techniques and especially the introduction of a number of nonradioactive nucleic acid probe modification and detection procedures in the 1980s [2] that the technique came into general use. Indeed, nonradioactive labeled probes offer advantages over conventional isotope labeled probes, as they produce higher resolution of the hybridization sites and lower background interference. Moreover, signals can be detected in only a few hours compared to several days or even weeks of exposure time. Over the past few years, an overwhelming number of papers covering different applications of the technique has appeared [3]. Nonradioactive in situ hybridization and detection has made chromosomal localization of specific sequences on human chromosomes possible [4, 5], Moreover, nonradioactive in situ hybridization has also been used to identify chromosomes [6], to detect chromosomal abnormalities [7], to study the spatial organization of genes in interphase nuclei [8] and to investigate the mechanisms of gene amplification [9]. Because nucleic acid probe can be labeled with a variety of different nonradioactive haptens, each of which can be visualized with an independent detection system, it is now even possible to detect two [10, 11] or three [12, 13] DNA sequences simultaneously. Further optimization of this multicolored in situ hybridization in terms of compatible probe labels, fluorochromes and optical filters is needed. Unfortunately, such progress and practical applications have been restricted essentially to cytogenetic studies of human and other mammalian systems. The development of in situ hybridization strategies for gene mapping as well as for other applications in plants has lagged behind that for animal chromosome systems. The major factor contributing to the slow development of this technique in plant cytogenetics is the difficulty of obtaining high quality mitotic metaphase chromosomes free of cell wall material. Moreover, the highly condensed nature of plant chromosomes, as well as the lack of marker chromosomes and reproducible banding procedures, makes karyotyp-ing extremely difficult.

Plant Cytogenetics

Books reviewed in this Article: W. Bergmann (ed.), Nutritional Disorders of Plants — Development, Visual and Analytical Diagnosis K.S. Labana, S.S. Banga and Banga (eds.), Breeding Oilseed Brassicas Singh, Ram J., Plant Cytogenetics Jørgensen, J. Helms (ed.), Integrated Control of Cereal Mildews: Virulence Patterns and Their Change Lea, Peter J., and Richard C. Leegood (eds.), Plant Biochemistry and Molecular Biology Arditti, J., and R. Ernst: Micropropagation of Orchids

Plant Breeding and Cytogenetics.

Despite its small nuclear genome and minute chromosomes, the model plant Arabidopsis thaliana has become a well established model in the field of plant cytogenetics. Since 2000, most cytogenetic and epigenetic approaches have been developed for this species and its congeners. In this chapter, we describe two step-by-step protocols for preparing chromosome preparations from root tip meristems and generative organ tissues of A. thaliana. Although both protocols have been optimized for A. thaliana, the detailed procedures are equally applicable to other members of the genus Arabidopsis as well as other crucifer species (Brassicaceae). © 2016 by John Wiley & Sons, Inc.

Cytogenetics was established based on the "Chromosome theory of inheritance", proposed by Boveri and Sutton and evidenced by Morgan's lab in early stage of the 20 th centrary. With rapid development of related research areas, especially molecular genetics, cytogenetics developed from traditional into a new era, molecular cytogenetics in late 1960s. Featured by an established technique named DNA in situ hybridization (ISH), molecular cytogenetics has been applied in various research areas. ISH provids vivid and straightforward figures showing the virtual presence of DNA, RNA or proteins. In combination with genomics and cell biology tools, ISH and derived techniques have been widely used in studies of the origin, evolution, domestication of human, animal and plant, as well as wide hybridization and chromosome engineering. The physical location and order of DNA sequences revealed by ISH enables the detection of chromosomal re-arrangments among related species and gaps of assembled genome sequences. In addition, ISH using RNA or protein probes can reveal the location and quantification of transcripted RNA or translated protein. Since the 1970s, scientists from universities or institutes belonging to the Jiangsu Society of Genetics have initiated cytogenetics researches using various plant species. In recent years, research platforms for molecular cytogenetics have also been well established in Nanjing Agricultural University, Yangzhou University, Nanjing Forestry University, Jiangsu Xuhuai Academy of Agricultural Sciences, and Jiangsu Normal University. The application of molecular cytogenetics in plant evolution, wide hybridization, chromosome engineering, chromosome biology, genomics has been successful. Significant progresses have been achieved, both in basic and applied researches. In this paper, we will review main research progresses of plant cytogenetics in Jiangsu province, and discuss the potential development of this research area.