When hydroponically cultured lettuce seedlings are transferred from a medium at pH 6.0 to another one at pH 4.0, the formation of root hairs takes place. Previous studies have revealed that this low-pH-induced root hair formation requires a phytochrome-mediated light signal and randomization of the existing transverse cortical microtubule (CMT) arrays in the future hair-forming cells, which occurs before the bulge formation. Here, we investigated the relationship between these two requisites, i.e., whether phytochrome is involved in CMT randomization. To test this, seedlings were cultured throughout in the dark except the various periods and timings of red (R) or far-red (FR) light irradiation. In seedlings that had already been transferred to pH 4.0 medium, R light irradiation induced CMT randomization, whereas neither FR light irradiation nor continuous dark culturing instead of R light irradiation did. R light irradiation during the pre-culture at pH 6.0 also caused CMT randomization later when the seedlings were transferred to pH 4.0 medium. Irradiation of FR light immediately after the R light irradiation suppressed the R light-induced CMT randomization, regardless of whether R/FR irradiation was carried out before or after the transfer of seedlings to pH 4.0 medium. The efficacy of R light and R/FR photo-reversibility in the induction of CMT randomization proves that the light signal needed for CMT randomization was indeed mediated by phytochrome, during lettuce root hair formation.

Meiotic studies were carried out on 26 accessions for the evaluation of genetic diversity of Cyanotis cristata and C. vaga from various geographical areas of Una, Hamirpur, Kangra, Palampur, Sirmaur (Himachal Pradesh), Dehradun and Mussoorie (Uttrakhand) in India. All accessions were diploids with n=12. The meiosis was found to be mostly normal in both species. In C. cristata, some meiotic cells showed abnormal figures such as early disjunction, bridges, laggards, interbivalent connections, vagrant or unoriented bivalents. All accessions of C. vaga showed normal meiotic course with low frequency of interbivalent connections. These species showed high pollen fertility in spite of presence of some meiotic abnormalities.

Cytological investigations have been carried out in one wild accession of Anemone rupicola Cambess. collected from the cold deserts of Ladakh division of Jammu & Kashmir India. The chromosome count of 2n=16 was observed and is the first chromosome record for the species from India. Meiotic analysis showed structural heterozygosity for reciprocal translocations as indicated by the presence of multiple associations of four chromosomes. Detailed meiotic analysis showed Pollen Mother Cells (PMCs) with laggards (32.87%) and chromatin bridges (21.91%) at anaphases I, II (A I, II)/telophases I, II (T I, T II), leading to abnormal microsporogenesis. Consequently, abnormal sporads such as triads (37.14%) and polyads (28.57%) were formed. Structural heterozygosis and other associated abnormal meiotic irregularities in the species showed considerable pollen sterility (32.20%) which could be attributed to the presence of ring/chain shaped multivalent in PMCs.

It is widely accepted that a symbiosis involving a cyanobacterium with a peptidoglycan wall led to the origin of plastids of photosynthetic eukaryotes. Recently, we visualized plastid peptidoglycan in a moss species by epifluorescence microscopy using metabolic labeling with click chemistry. In the present study, we applied the same method to visualize plastid peptidoglycan in the filamentous charophyte alga Klebsormidium nitens, as its genome contains a set of genes capable of synthesizing peptidoglycan. To visualize peptidoglycan, the generation of D-alanyl-D-alanine (DA-DA) must be blocked to allow the incorporation of ethynyl-DA-DA (EDA-DA) into peptidoglycan. Because a gene-targeting technique has not been established for K. nitens, we used D-cycloserine, which is an inhibitor of D-Ala : D-Ala ligase. Treatment with 500 µM D-cycloserine arrested cell division and inhibited chloroplast division. The addition of 1 mM EDA-DA restored the rate of cell division to that observed in medium without D-cycloserine, suggesting that EDA-DA can integrate plastid peptidoglycan instead of DA-DA, thus restarting chloroplast division and therefore cell division. Subsequently, cells were subjected to click chemistry to attach an azide-modified Alexa 488 fluorophore to the EDA-DA probe. Microscopic observation indicated that both sides of chloroplasts have strong Alexa 488 fluorescence. Under conditions of no cell growth, the chloroplast peptidoglycan that existed prior to treatment with D-cycloserine might remain in the cell. Therefore, the fluorescence must be indicative of the location of newly synthesized peptidoglycan. Our observations suggest that chloroplast peptidoglycan is built along the chloroplast division plane and serves as an essential system for chloroplast division in K. nitens.

Plant protoplasts are valuable in biotechnology, enabling from plantlet regeneration to the determination of gene function. Viability tests are required in protoplast applications to determine the rate of viable cells, allowing to decide on the most adequate isolation and purification procedures and to verify whether sufficient cells are available for subsequent steps. Fluorescence microscopy is usually employed for this purpose. However, obstacles have pointed out: the long time required to count a relatively small number of protoplasts, cell clumps preventing their observation, and the observer’s subjective visual perception of the fluorescence. Therefore, this study aimed to establish procedures for the viability test adapted for flow cytometry (FCM), Muse cell analyzer (Muse) and Comet Assay (CA). Capsicum annuum L. was chosen as a study species based on the recalcitrant morphogenic nature of its protoplasts. After isolation and purification, the FCM and Muse allowed assessing large numbers of protoplasts, and the CA the protoplast nuclei within a short period. Using the adjusted procedures, various cytological characters could be evidenced by FCM and Muse, and different levels of genomic damage evaluated by CA, allowing the discrimination and measurement of the viable protoplasts. Considering these results, the present study introduces improved quantitative procedures for viability tests. Moreover, aiming at plantlet regeneration, distinct applications should employ to measure the protoplast viability and thus define the most adequate isolation and purification procedures. Contributing with this purpose, guidelines present here to adequate and standardize the laboratory conditions for protoplast viability test using FCM, Muse and CA.

Imaging techniques with split fluorescent proteins are based on the reconstruction of separated asymmetric protein fragments. Although each fragment alone has no fluorescence, a reconstructed protein after the association of its constituent fragments recovers its fluorescence. In the case of the split-GFP assay, GFP is divided between the 10th and 11th β sheets to generate two asymmetric fragments, GFP1–10 and GFP11. GFP11 is a small tag that minimally disturbs the localization and dynamics of a fused protein. Thus, the split fluorescent protein assay enables analyses of protein localization patterns, protein–protein interactions and the topology of membrane proteins.

Hymenocallis littoralis was previously reported various chromosome numbers of 2n=44, 46, 48, 49 and 68 by a conventional staining. In order to analyze these chromosomes in detail, karyotype analysis by the conventional staining and a fluorescence in situ hybridization (FISH) using probes of 45S and 5S rDNA, and telomere repeats of human-type (TTAGGG) n and Arabidopsis-type (TTTAGGG) n was performed on chromosomes of H. littoralis from Thailand. The plants have 26 chromosome pairs (2n=52), and this chromosome number was not consistent with any of the previous reports. The 45S rDNA signals were localized at the terminal region of short arms of two chromosome pairs. Heteromorphic signals of 5S rDNA were detected at the proximal region of the short arms of one long chromosome, and at the interstitial region of the long arm of one short chromosome. The signals of human-type telomere repeat were detected at all chromosome ends, but FISH with Arabidopsis-type telomere repeat showed no signal.

The karyotypes and NOR condition of the secondary constrictions of chromosome pair 1 in two populations of Crotalaria incana from the Atlantic and Pacific slopes of Mexico are described. Both populations exhibited the same chromosome number of 2n=14 and karyotypes which shared a secondary constriction on the short arm of chromosome pair 1. Differences were found in the position of the centromere on chromosome pair 5 and in the size of the proximal region of the short arm of chromosome pair 1. In addition, marked differences were observed in total haploid chromosome length (THC) and mean chromosome size (MC), without this affecting asymmetry indexes. Atlantic population exhibited chromosome fragments and fragile sites at prometaphase. These data revealed differences in karyotypes between two populations.

Nucleolar organizer regions (NORs) and karyotype in the black lancer, Bagrichthys majusculus from Nakhon Phranom and Sing Buri provinces, Thailand, were investigated. The mitotic chromosome preparations were directly obtained from kidney tissues of eight male and eight female specimens. Conventional and Ag-NOR staining techniques were applied to the chromosomes. The chromosomes number of B. majusculus was 2n=50 and the fundamental number (NF) of all specimens was 96. The karyotype consists of six large metacentric, six large submetacentric, two large acrocentric, eight medium metacentric, 12 medium submetacentric, two medium acrocentric, six small metacentric, four small submetacentric and four small telocentric chromosomes. Sex chromosome was not observed. Moreover, the interstitial NORs were clearly observed at the long arm of the metacentric chromosome pair 1. The result revealed that a heteromorphic NOR type was appeared in one male and one female fish and other fish have homozygous NOR in the pair 1 chromosomes.

Triploid cytotypes of Polystichum squarrosum have been found from India. All accessions shared the same cytological status of 2n=123, the first ever record against the earlier reported diploid (2n=82) and tetraploid (2n=164) cytotypes from India. Individuals depicted highly abnormal meiotic behavior characterized by presence of univalents beside bivalents, unoriented chromosomes at metaphase I (MI), laggards, chromatin bridges at anaphase and telophase. Cytomixis at tetrad showing nuclear fusion between sporads is another noticeable phenomenon in this species. Triads, polyads and micronuclei at tetrad stage consequently lead to low spore fertility and formation of heterogenous spores. B-chromosomes were found in few cells in one of the accessions.