The Wada Memorial Award was created in 2011 to encourage the submission of high-quality papers to Cytologia. Coincidentally, the first to fourth holders of the award were experts in karyotype and chromosome analyses among the three regions of the Indomalayan biogeographic realm. Most of Indomalaya was originally covered by tropical and subtropical moist broadleaf forests, with high biodiversity that is currently at risk due to various anthropogenic influences. Karyotype and chromosome analyses provide an essential foundation for maintaining biodiversity through conservation; therefore, in this paper, we focus recent their contributions of the early Wada Memorial Award winners to the three regions of Indomalayan

The meiotic chromosome numbers of six Carex species in three sections are counted; C. miyabei Franch. (n=42_II) in sect. Carex, C. breviculmis R. Br. (n=31_II, 32_II, 33_II), C. polyschoena H. Lév. & Vaniot (n=36_II, 37_II), C. sabynensis Less. ex Kunth (n=27_II, 28_II) in sect. Mitratae Kük., and C. heterolepis Bunge (n=37_II, 38_II, 40_II) and C. forficula Franch. & Sav. (n=25_II, 27_II) in sect. Phacocystis Dumort. The chromosome counts for C. miyabei, C. heterolepis, and C. forficula from Korean populations are verified for the first time. Considered with previous reports, all the species exhibit variations in chromosome number from n=4 to n=36. Contiguous variation in chromosome numbers and non-constricted meiotic chromosomes with holocentromeric nature suggest that aneuploidy and/or agmatoploidy and/or symploidy should have played an important role in the sections.

Lamiaceae is a requisite depository of many important medicinal herbs, distributed all over the world. The detailed meiotic studies, firstly are helpful in solving taxonomic and evolutionary enigmas and secondly provide best chemotypes for further exploration and conservatory purposes. Male meiosis in 29 species of 54 populations belonging to the 18 genera of Lamiaceae has been undertaken for the first time from Sirmaur district of Himachal Pradesh falling in Western Himalayas. Euploid cytotypes are reported in Ajuga parviflora (n=8x=32), Nepeta eryostachys (n=4x=18) and N. graciliflora (n=4x=18). Besides, B-chromosomes in previously known cytotypes of five species, A. bracteosa (n=32+0-2B), Leucas lanata (n=11+0-1B), Lycopus europeus (n=11+0-1B), Phlomis bracteosa (n=11+0-1B) and Vitex negundo (n=16+0-1B), are reported for the first time. A substantial amount of meiotic abnormalities in the form of chromatin stickiness, cytomixis, chromatin bridges and laggards are reported in the presently worked out population that leads to the high variability in the microsporogenesis, pollen grain size heterogeneity and ultimately, reduce pollen viability. Only, the populations with B-chromosome had a low or negligible frequency of meiotic abnormalities.

Intraspecific euploid cytotypes (2n=18, 36) were found in the wild plants of Brachyactis pubescens in North West Himalayas from the hills of Ladakh division of Jammu and Kashmir and Solang Valley, Kullu district, Himachal Pradesh. The plants with each ploidy level showed regular bivalent formation, equal segregation of chromosomes during anaphases and normal sporads. The 2x plants were found growing at higher elevations in the Kashmir Himalayas and adjoining hills of Afghanistan while the 4x plants grow at lower elevations in the Solang Valley, Himachal Pradesh. The 4x and 2x plants could be differentiated in various morphometric parameters. The 4x plants were noticed to be robust and much taller and show gigantism in leaf and floral characters besides depicting the increase in the size of trichomes, stomata and pollen grains. Analysis of meiotic chromosome pairing in the tetraploid taxa indicates towards its allopolyploid nature.

Chronic obstructive pulmonary disease (COPD), characterized by long-term breathing problem and poor airflow, is the fourth cause of death in the United States. Although previous studies have provided insights into the effects of genetic factors on COPD, the molecular mechanism is still unknown. Here, we proposed a network and pathway-based method for prioritization and analysis of COPD-related genes. In brief, we firstly obtained COPD-related single nucleotide polymorphisms (SNPs) from dbGaP and COPD as well as normal lung tissues gene expression profiles from Gene Expression Omnibus (GEO). Combined with protein–protein interaction pairs, the SNPs and gene expression profiles were imported into dmGWAS, a R package designed for subnetwork searching, to identify COPD-specific module. What’s more, we performed functional analysis for genes in COPD-specific module with the combination of WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) and KEGG Orthology Based Annotation System (KOBAS). A COPD-specific network module containing 812 gene nodes and 2640 interactions was obtained. While, 450 of the 812 genes were annotated by WebGestalt and/or KOBAS, which were considered as reliable COPD-related genes. The annotated genes were found to be significantly associated with immune and signal transduction processes, which is consistent with the development of COPD. Finally, 427 of the 450 annotated genes formed 1389 interaction pairs, which might contribute COPD progression. This study should be helpful for understanding COPD mechanisms and its targeted therapy.

The family Commelinaceae shows a lot of morphological and cytological diversity. Chromosome numbers and male meiosis of 15 populations of 12 species under four genera i.e., Amischophacelus, Commelina, Cyanotis and Murdannia of Commelinaceae have been studied from Rajasthan, India including the first chromosome report of Commelina forskaolii Vahl (2n=90) in the world. It includes new euploid cytotypes in six species i.e., C. attenuata (2n=24), C. diffusa (2n=120), C. hasskarlii (2n=120), C. paludosa (2n=30), Cyanotis cristata (2n=22) and Murdannia nudiflora (2n=80) besides new chromosome reports from Rajasthan in three other species i.e., Amischophacelus axillaris (2n=20), Co. benghalensis (2n=22) and Cy. fasciculata (2n=22). Meiosis is regular in seven species but has a low frequency of meiotic anomalies like laggards, chromatin bridges and stickiness in five species (C. albescens, C. benghalensis, C. forskaolii, C. suffruticosa and M. nudiflora).

Karyotype and nucleolar organizer regions (NORs) were studied in the longfin carp (Labiobarbus leptocheilus) from Mekong Basin of Thailand. The mitotic chromosomes were directly prepared from kidney cells from five male and five female fishes and stained by conventional staining and Ag-NOR banding. The chromosome number was 2n=50 and the fundamental number (number of the chromosome arms, NF) was 86 in both male and female fishes. The karyotype was composed of six large metacentric, eight large acrocentric, six large telocentric, four medium metacentric, six medium submetacentric, six medium acrocentric, four medium telocentric, four small metacentric, two small acrocentric and four small telocentric chromosomes. The Ag-NOR banding showed a single pair of Ag-NORs adjacent to the telomeric position of the short arm of the chromosome pair 8.

Plants can utilize soil nitrogen (N) as ammonium-N (NH₄⁺-N) or nitrate-N (NO₃⁻-N). Ammonium nitrate, an inorganic N fertilizer, having both forms of N, when added to the soil, causes an immediate change in the soil N dynamics. The ratio of ammonium to nitrate keeps changing and influences the soil pH and thus the rhizosphere environment sometimes making it toxic. To investigate the genotoxicity of ammonium nitrate fertilizer; Allium cepa L. was used as a model plant. A pot experiment was designed in which ammonium nitrate fertilizer was added to soil at the rate of 0.0, 0.2, 0.5, 0.8, 1 and 2 mg N g⁻¹. Inorganic N nutrients in the form of NH₄⁺-N and NO₃⁻-N of soil samples and their ratio were investigated at 1, 7, 15, 30 and 45 days after treatment (DAT). Allium bulbs were also planted in each pot and three replicates were maintained for each dose of ammonium nitrate at 1, 7, 15, 30 and 45 DAT. Roots of Allium bulbs were harvested on the day 3 of sowing and fixed for cytological analysis. The result showed mitodepressive activity at higher concentrations of fertilizer, along with chromosomal anomalies which revealed the genotoxicity of ammonium nitrate. A significant positive correlation was observed between fertilizer concentrations and NH₄⁺ content of treated samples; similarly NO₃⁻ content also showed a positive correlation. The change in the ratio of NH₄⁺ to NO₃⁻ with respect to time and treatments are suggested as the possible reason for the disturbance in nuclear and chromosomal behavior.

The present study was designed to determine the allelopathy effects of aqueous extracts of leaves of two medicinal plants of Sapindus mukorossi Gaertn and Leucaena leucocephala (Lam.) de Wit. on seed germination, radicle length, mitotic index and chromosomal aberrations in two verities of vegetable crop pea (Pisum sativum), the most popular in India. The results of the present experiments revealed that the extracts of the two medial plants have an inhibitory effect on seed germination, radicle growth, mitotic index and chromosomal aberrations in crop pea owing an occurrence of allelochemicals.

Tecomella undulata (common name Rohida) is a species of monotypic genus in Bignoniaceae found growing in arid regions of the world. Plant is known for its ornamental and medicinal use. The plant is a small or medium sized tree bearing flowers in three different colors and is presently included in the category of endangered plants in India. Three morphotypes based on flower color have been recognized in it i.e., red flowered Rohida (RFR), orange flowered Rohida (OFR) and yellow flowered Rohida (YFR). The plants were investigated cytogenetically and at a molecular level to detect the variability. Cytological investigation showed that all the plants possess the chromosome number of n=18 irrespective of the morphotype studied with high pollen fertility. The molecular diversity studies were carried through a random amplification of polymorphic DNA (RAPD) analysis. Amplification of genomic DNA of ten accessions of T. undulata using six RAPD primers yielded 87 fragments out of which 75 were polymorphic. The RAPD bands obtained from six primers were finally used to infer genetic distance between the morphotypes. OFR accessions had higher genetic diversity with the Nei’s expected heterozygosity (h)=0.266, Shannon’s information index (I)=0.388 and percentage polymorphism of 62.52%. The present study concludes that though the plant showed same chromosome number, intraspecific variability exists in T. undulata that has a molecular genetic basis. The genetic information can be used for proper conservation of the plant besides being used as a tool to select plants for breeding purpose.

We previously established an experimental system for efficient pathogenic signal induced-cell death using tobacco BY-2 cells and culture filtrate of a plant pathogenic bacterium, Erwinia carotovora. Using this experimental system, cytoskeletal and vacuolar changes during the process of filtrate-induced cell death were characterized in detail. However, the initial events induced by the filtrate were largely unknown. In this study, reactive oxygen species (ROS) production and endocytosis stimulation were examined in BY-2 cells treated with the filtrate of E. carotovora. Transient ROS production within 2 min was observed after the filtrate treatment. Treatment with the Ca²⁺ chelator BAPTA or the protein kinase inhibitor K252a significantly inhibited ROS production in a dose-dependent manner, suggesting that ROS production depends on Ca²⁺ influx or protein phosphorylation. In addition, internalization of the endocytic marker FM4-64 was promoted by filtrate treatment within 30 min, suggesting that the filtrate treatment stimulated endocytosis. These results showed that ROS production and subsequent endocytosis stimulation are the initial events induced by culture filtrate of E. carotovora in BY-2 cells.

The purpose of this study is to reveal diploid chromosome number, karyotype and chromosomal banding properties with C-banding and Ag-staining in Cobitis phrygica Battalgazi, 1944 and C. simplicispina Hanko, 1925 from Turkey. Metaphase chromosomes were obtained from kidney cells. Both species had a same diploid chromosome number of 2n=50. Karyotypes were composed of four pairs of metacentric, four pairs of submetacentric and 17 pairs of subtelo-acrocentric chromosomes in C. phrygica, and eight pairs of metacentric, eight pairs of submetacentric and nine pairs of subtelo-acrocentric chromosomes in C. simplicispina. Fundamental arm numbers were calculated as 66 in C. phrygica and as 82 in C. simplicispina. C-bands were observed on the pericentromeric regions of most chromosomes in both species. Nucleolus organizer regions (NORs) were determined on one pair of chromosomes in both species.

Convolutional neural networks (CNNs) are used for various data analyses and resemble the human brain cognition system. CNN algorithms are composed of multiple layers of convolution and pooling layers. Recent studies have shown that applying CNN algorithms to classify biological images are feasible. The alga, which has various morphological features, is the one of the interesting targets to classify using CNN. As an example, we targeted unicellular Cyanidioschyzon merolae. C. merolae is a primitive red alga that is not only used for studying organelles but also to proceed the study of the production of lipids. Measuring the division rate requires the classification of the interphase and mitotic phase of C. merolae. In this study, we constructed an automatic classifier for interphase and mitotic C. merolae. By tuning the hyper-parameter, number of layers, and number of insertions of dropout functions, we classified the interphase and mitotic C. merolae with the high accuracy of 92%.

Two dwarf mutant lines of Coriandrum sativum L. (Apiaceae) designated as mut. I and mut. II are screened at M₃ to possess concomitant desynaptic behavior of chromosomes. The mutants are raised following seed treatments with cadmium sulfide nanoparticles (CdS-NPs) at 0.50 µg mL⁻¹ for 4 h duration (mut. I) and 200 Gy gamma irradiations (mut. II). Both the desynaptic mutants are ‘medium strong’ type demonstrating the enhanced number of univalent per cell and reduced chiasma frequency per nucleus at diplotene–diakinesis and metaphase I (M I). The univalents in the mutants are random in distribution as well as polar and equatorial orientations. Mean chromosome association per cell at M I is 10.63_II+0.74_I in control, 9.55_II+2.91_I in mut. I and 9.64_II+2.72_I in mut. II plant types. The mutant meiocytes show variable types of associations at M I including 22_I formation; although, the predominant association is 11_II. Both the mutants demonstrate different types of irregular chromosome separations and bridge formation with or without a fragment in anaphase I (A I) and anaphase II (A II) cells. Fertility of pollen grains is 94.47% in control plants and is reduced in the mutants. The potentiality of CdS-NPs in inducing cytogenetical mutant like desynapsis alike to gamma irradiations is highlighted.

Chromosome counts including analyses of male meiosis and pollen fertility have been made on 36 accessions of 10 Lactuca species collected from the western Himalayas. L. orientalis (2n=18) and L. undulata (2n=18) have been counted chromosomally for the first time from India and the counts in other species confirmed previous counts. The study also adds the existence of intraspecific diploid cytotype for L. orientalis. B-chromosomes have been reported for the first time in the accession of L. hastata. Cytomixis has been detected in some accessions of L. dolicophylla, L. macrorhiza and L. scariola. Consequent to cytomixis, the accessions were depicted some pollen sterility. Based on compiled chromosomal information it is apparent that dysploidy in conjugation with polyploidy seems to play an important role in the evolution of genus the Lactuca seems to be polybasic (x=5, 8, 9, 17), and x=8 and x=9 seem to be the most common and favored base numbers.

The somatic chromosome numbers and karyotypes were examined in seven species of genus Centaurea L. distributed in Turkey. The chromosome numbers were determined as 2n=2x=16 in C. behen L. and C. solstitialis L.; 2n=2x=18 in C. cynarocephala Wagenitz and C. agregata Fisch. & Mey. ex DC. ssp. agregata; 2n=2x=20 in C. iberica Trev. ex Sprengel; 2n=2x=26 in C. balsamita Lam.; 2n=4x=36 in C. virgata Lam. All of the chromosomes had centromeres at median point (M), median region (m), and submedian region (sm). Satellite chromosomes (sat-chromosomes) were observed only in C. virgata. The findings for each of the analyzed taxa are compared each other and with the results of previous studies.

We present the results of cytogenetic analysis of seven young and long-term cultured embryogenic cell lines (CLs) of Larix sibirica obtained by somatic embryogenesis. All CLs were found to be mixoploid. Three CLs (1, 2 and 6 years of cultivation) were the most stable cytogenetically with 91–96% cells having diploid chromosome number 2n=24, typical for the species. These CLs can be successfully used for the development of planting material and in clonal plantation forestry. At the same time, some embryogenic CLs were shown to possess various chromosome and genome mutations. We found two aneuploidy proliferating CLs (2n=25 and 28) capable of somatic embryo formation. One of the CLs analyzed in a current study was found to be cytogenetically unstable with chromosome numbers ranging from 24 to 35 and containing one-two micronuclei in the interphase cells. Fluorescent in situ hybridization (FISH) and morphometric analyses of karyotypes of these embryogenic CLs allow an assumption that the trisomy for one or several chromosomes is the most widespread type of genomic disturbances in embryogenic CLs of L. sibirica.

Dry seeds of Nigella sativa L. (Ranunculaceae) are exposed to azo-dyes [methyl orange (MO) and malachite green (MG)] and heavy metals [cadmium (Cd) and lead (Pb)] following impregnation of a filter paper for 12 days at an interval of 48 h. Cytotoxicity on mitotic index, types, and frequency of cytological abnormalities and apoptotic cell death induced by the test materials are assessed in relation to control. All the studied environmental pollutants were found to reduce germination and growth, inhibit cell division, enhance cytological aberrations and cause apoptotic cell death. Fluorescence activated cell sorting (FACS) analyses not only reveal apoptosis but also induced metabolic peak shifting and polyploid cell formation at late G₂ phase. Results suggest that heavy metals (Pb＞Cd) show more toxicity than azo-dyes (MG＞MO). The objective of the investigation is to predict safety dose for commercial exploitation of the studied azo-dyes and heavy metals. From the standpoint of which the employed low doses of the studied environmental pollutants are found to induce cytotoxicity affecting germination and growth in seedlings of N. sativa.

Structome analysis, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, is a useful tool for identification of unknown microorganisms that cannot be cultured. In 2012, we discovered a unique microorganism with a cell structure intermediate between those of prokaryotes and eukaryotes from the deep sea off the coast of Japan and named it Parakaryon myojinensis. We also reported another unique bacterium found in the same place that we named as Myojin spiral bacteria. Here, we report the third unique bacteria we discovered by structome analysis and 3D reconstruction using serial ultrathin sectioning of freeze-substituted specimens from the same place. The bacteria showed elongated flattened cell bodies with uneven surfaces. The cells consisted of outer amorphous materials, cell wall, cytoplasmic membrane, ribosomes, fibrous materials, and vacuoles. They had a total length of 1.82±0.40 µm, a total volume of 0.37±0.09 µm³, and had 1150±370 ribosomes within a cell; the density of the ribosomes in the cytoplasm was 312±41 per 0.1fL. Each bacterium showed different shapes but appears to belong to a single species because they have similar size and volume, have similar internal structure, inhabit a confined area, and have similar ribosome density in the cytoplasm. We named it the ‘Myojin amorphous bacteria’ after the location of discovery and its morphology. This is the first report on the existence of amorphous bacteria.