The various behaviors of cells are closely related to the temporal and spatial dynamics of chromatin. Live cell imaging techniques for chromatin can provide insight into gene expression at the three dimension of a nucleus and offer the novel knowledge in gene regulation for medical diagnosis, crop improvement, and environmental remediation. However, traditional chromatin imaging techniques that rely on the insertion of a target gene are unable to directly analyze the chromatin dynamics in living cell nuclei. CRISPR-based live imaging, with its high accuracy, speed, and low perturbation, has emerged as a reliable tool for high-resolution imaging of the specific chromatin in the nucleus. This review paper introduces the benefits of CRISPR-based live imaging for chromatin dynamics, classifies and analyzes various successful schemes used to improve the signal-to-noise ratio of this method, and discusses future trends in the field.
Chromosomal characteristics of Dasylirion acrotrichum (Asparagaceae), Flourensia resinosa, (Asteraceae), Tecoma stans (Bignoniaceae), and Citharexylum brachyanthum (Verbenaceae), common elements of the Mexican xerophile scrub are recorded for the first time. The spreading and air-drying technique in root meristems allowed for obtaining chromosome preparations. Chromosomal parameters such as the diploid number (2n), karyotype formula (KF), total haploid chromosome length (THL), average chromosome size (AC), and asymmetry index (TF%) of the analyzed species were obtained, except in C. brachyanthum where neither KF nor TF%. The karyotype formulae of D. acrotrichum (13m+6sm), F. resinosa (11m+7sm), and T. stans (14m+3m+1stsat) represent the first cytogenetic records in Mexican populations. Differences in KF, AC, and position of the satellites concerning other previously analyzed cogenerics show that speciation involves changes in the size and shape of the chromosomes. The diploid numbers confirm basic numbers (x) already recorded, but a 2n=104 observed in C. brachyanthum and the same number in C. myrianthum from Brazil could modify x=9 in favor of x=13, consistent with karyological variation in Verbenaceae where hybridization and polyploidy, among other numerical variation mechanisms, play an important role in chromosomal differentiation. The taxa studied showed satellites in the short arms of chromosomes with displaced centromere. These data are taxonomically useful and could be associated in part with the geographic distribution of the species.
Cytogenetical studies were conducted on Ledebouria botryoides. We reported 2n=60 (15m+10sm+5st) chromosomes. The karyotype was asymmetrical and fell into the 4B category of Stebbins’ asymmetry classes. Two groups of chromosomes were observed, i.e. 15 pairs of long chromosomes (2.82–6.65 µm) and 15 pairs of short chromosomes (1.67–2.80 µm). Two chromosome pairs had a satellite on the short arms. The meiotic course was found to be normal with n=30 bivalents.
Four rare species of genus Alocasia in Thailand were cytologically studied. The somatic chromosome number and karyotypes of four rare species are 2n=28=12m+12sm+4st (A. heterophylla), 2n=28=16m+8sm+4st (A. hypoleuca), 2n=56=34m+12sm+10st (A. princeps ‘Purple Cloak’) and 2n=26=12m+10sm+4st (A. nebula), respectively. The somatic chromosome numbers of all species were reported for the first time except A. princeps ‘Purple Cloak.’ The NF and karyotype of all species were reported for the first time. The cytogenetic data in this study can be used for the identification of each species.
In higher groups of plants, the somatic cells are conventionally used for karyomorphological studies, which contain a diploid (2n) set of chromosomes. Similar to diploid sporophytic cells, a chromosomal study in haploid (n) gametophytic cells is also necessary as it unfolds the karyomorphological status of germ cells. The mature male gametophytic plant body (i.e., pollen grain) is only two- or three-celled structures. This study focused on the first mitotic division in a microspore, popularly known as ‘pollen mitosis-I.’ Allium cepa L. and Aloe vera L. are two model species for karyomorphological study. Microspores of these species have been stained with 2.0% acetocarmine. The present finding reveals that the cell division in microspores of these two species is completely normal without any significant irregularities. As expected, the chromosome number of the microspores of Allium cepa is n=8 and Aloe vera is n=7, exactly half the number of their sporophytic plants. The haploid karyotype of Allium cepa is the symmetric type with karyotype formula ‘6m+2sm,’ whereas that of Aloe vera is between symmetric and asymmetric types with karyotype formula ‘3sm+4st.’ This study not only enriches the chromosomal database of gametophytes of Allium cepa and Aloe vera, but also will encourage further research on this extremely neglected topic—chromosome study in the gametophytic generation of flowering plants. As Allium cepa and Aloe vera are easily available and model species for karyomorphological study, and with two contrast karyotypes (i.e., symmetric and asymmetric), this article also helps numerous school/college teachers to teach their students about the structure and behavior of gametophytic chromosomes.
Sexual reproduction in angiosperms is a complex and precise process of regulation, which includes the pollen tube guidance, double fertilization, and the seed development process. The previous report identified that for seed development, there is a period of preparation for fertilization (between pollen tube guidance and double fertilization) that is called pollen-tube-dependent ovule enlargement morphology (POEM). It has been shown that the pollen tube content (PTC) plays a crucial role in the enlargement of ovules and the initiation of seed coat formation. However, we did not investigate the potential of endosperm proliferation in autonomous mutants at a later stage. Here, we investigated this phenomenon using vanillin staining and transparent experiments to examine the manner, in which the PTC affects the potential of endosperm formation. Interestingly, the PTC increased the number of endosperm nuclei without fertilization equally and synchronously in mea and fis2 ovules. This finding might help improve the study of apomixes and our understanding of how the molecular mechanisms that regulate this phenomenon will contribute to plant reproductive science in the future.
Cell walls in the giant-celled green alga Valonia are composed of multiple lamellae and designated as “crossed-fibril” type wherein each lamella contains cellulose microfibrils (CMFs) arranged in a uniform orientation. Outer, older lamellae are thinner than inner, younger lamellae, probably because the former are extended during cell growth more so than the latter without deposition of new cell wall materials. If this hypothesis is correct, the density of CMFs in the outer lamellae should be lower than that in the inner lamellae. In the present study, the density of CMFs in the outermost lamellae of the cell wall, in various sizes of Valonia utricularis growing cells, was calculated using images obtained with an atomic force microscope, and the densities were compared among the cells of different sizes. The CMF density ranged from 0.5 to 4.0 µm−1, which is lower than that previously recorded for the innermost lamellae (more than 10 µm−1). Moreover, the CMF density was negatively correlated with the cell size indicating that spacing between neighboring CMFs was increased by the cell surface extension due to cell growth. These findings may support the abovementioned hypothesis that the outer cell wall lamellae are extended by cell growth without the deposition of new CMFs.
Fluorescence chromosome bandings with CMA- and DAPI- were analyzed in six wild Corchorus species to find out the diversity in the number and location of AT- and GC-rich sequences. These six wild Corchorus species had variation in somatic chromosome numbers viz. C. brevicornatus (2n=14), C. septentrionalis (2n=14), C. urticifolius (2n=14), C. hirtus (2n=28), C. siliquosus (2n=28) and C. pinnatipartitus (2n=78). A wide range of CMA- and DAPI-positive bands were found in the metaphase chromosomes of six wild Corchorus species. A total number of 35 and 32 terminal CMA- and DAPI-bands were generated which recommended the tendency of accumulation of GC- and AT-rich sequences at the terminal region of chromosomes, respectively. In some species, CMA-banded satellites were extended in prometaphase while contracted at the terminal region of the short arm in the metaphase stage. Reshuffling of chromosomal segments was detected following CMA- and DAPI-banding. After reversible fluorescent banding, C. brevicornatus, C. urticifolius and C. siliquosus had a few CMA-positive and DAPI- negative banded chromosomes which could be used as marker chromosomes. In addition, few chromosomes of C. pinnatipartitus could be used as markers due to their unique DAPI-banding pattern. Each species had its characteristic CMA- and DAPI-banding pattern.
Aloe trinervis C.S.Purohit, Kulloli & Suresh Kumar has only species described from India and has narrow distribution in the state of Rajasthan. A. trinervis is related to the A. vera (L.) Burm.f. but very distinct by the vegetative and reproductive characteristics. Thus, in the present investigation, somatic chromosome number and karyotype analysis are reported for the first time in A. trinervis. The karyotype of the species is the 1C asymmetric category of Stebbins classification and the karyotypic formula is 2n=14=6sm+8st.
Paeonia (Paeoniaceae) has an ancient origin, unique genetic evolution, and an isolated taxonomic status. China is the genetic origin of the genus Paeonia and an area rich in wild Paeonia resources. However, since herbaceous peony is not as well studied as woody peony, especially some species that are currently endemic to China. One such species that is endemic to China is Paeonia mairei, which is valuable germplasm for evolution research and cultivar improvement. Despite this, the karyotype of P. mairei remains inconclusive. In this study, root tips of plants from eight populations of P. mairei were used to examine their ploidy and to assess karyotype diversity. The results indicate that all eight P. mairei populations were tetraploid (2n=4x=20=12m+4sm+4st) with a 2A karyotype. Karyotype asymmetry ranged from 60.61% for P6 (Liangfengya, Foping County) to 63.12% for P4 (Ningqiang County). The chromosome length ratio ranged from 1.7398 (Dongchuan District) to 1.9660 (Dayi County) while the AI ranged from 4.4535 (Ningqiang County) to 5.6085 (Foping County). These findings indicate that the population in Foping County is relatively evolved whereas that in Ningqiang County is relatively primitive. Cluster analysis of karyotypic parameters showed that P3 (Dayi County) and P7 (Miaogou, Foping County) populations differed greatly from the other six populations, which could be clustered into two geographic branches. Our findings provide a scientific basis for exploring origin and evolutionary characteristics of P. mairei.
The einkorn wheat (Triticum monococcum) strain KU104-2 is an early-flowering line that was generated by X-irradiation of the einkorn wheat strain KU104-1. The early-flowering KU104-2 heads about one month earlier than KU104-1 and has a deletion of the biological-clock-component gene PHYTOCLOCK 1/LUX ARRHYTHMO (PCL1/LUX). In this study, we characterize a newly induced mutant named late-heading 1 (lh1) that was generated by heavy-ion beam irradiation of KU104-2. This new mutant shows partial suppression of the early-flowering phenotype of KU104-2. Under field conditions, lh1 plants head about one week later than KU104-2; lh1 mutants show no other significant differences in agricultural traits such as plant height, spike length, and spikelet number per spike compared with KU104-2. Analysis of plant development in a growth chamber showed that lh1 mutants exhibit a late-flowering phenotype under short-day (SD) conditions, but not under long-day (LD) conditions. This late-flowering phenotype under SD conditions is associated with the down-regulation of the flowering promoter gene VERNALIZATION 1 (VRN1) in leaves of plants at the 5th-leaf stage of growth, i.e., at the late vegetative phase. Our findings suggest that the expression level of VRN1 in the vegetative phase under short-day conditions is important for the determination of flowering time in wheat.
Information on chromosomes and estimation of genome size could be applied to study the evolution and classification of a genus. In our study, the conventional pressing method was applied to prepare microscope slides for counting the chromosomes, and the chromosome numbers of all surveyed species were 2n=32, 34, 36, 38. The first-time determination of chromosome counts for 12 taxa and karyotypes for 16 taxa in Callicarpa were conducted. It exposed all the species with a higher ratio of m chromosomes and a lower ratio of sm chromosomes, and only C. macrophylla contained st chromosome pair. The minor differences between the karyotypes of all species revealed that the karyotype diversity of this genus evolved from a tiny variation of chromosome structure. Meanwhile, the genome size of 23 samples from 16 taxa of Callicarpa has been estimated by flow cytometry. The genome size ranged from 0.96 pg (C. pedunculata) to 1.26 pg (C. nudiflora), averaged out at 1.12 pg, and the variation of genome sizes was fine among the investigated species. Hence, it could also be referred from the viewpoints of cytology that Callicarpa was a group in Lamiaceae that evolved earlier and independently.
Previous chromosome information for 2n=28 is restricted to 17 species in four Asian Begonia sections Baryandra, Diploclinium, Petermannia and Reichenheimia. Here we report the first chromosome counts and karyotype analyses of 2n=28 for three species in two Asian sections Flocciferae and Parvibegonia, as well as those for 2n=56 of B. baliensis in sect. Platycentrum. The number of sections with 2n=28 reported is increased to six of 20 sections of Asian Begonia. Based on the karyotypes, the chromosome evolution for 2n=28 as well as 2n=56 in Asian Begonia is discussed.
Karyomorphological features of Aspidistra longifolia and A. subrotata from Thailand were here reported. The karyotype of A. longifolia is analyzed and confirmed as 2n=38=24m+2sm+12st in Doi Tung, Phu Hin Rong Kla National Park, and Nam Nao National Park. The tetraploid population of A. subrotata has been found in Phu Luang Wildlife Sanctuary, as the second tetraploid population reported for this species so far and the westernmost known record.