2C DNA Research Articles

Germination and the Early Stages of Seedling Development in Brachypodium distachyon.

Successful germination and seedling development are crucial steps in the growth of a new plant. In this study, we investigated the course of the cell cycle during germination in relation to grain hydration in the model grass Brachypodium distachyon (Brachypodium) for the first time. Flow cytometry was performed to monitor the cell cycle progression during germination and to estimate DNA content in embryo tissues. The analyses of whole zygotic embryos revealed that the relative DNA content was 2C, 4C, 8C, and 16C. Endoreplicated nuclei were detected in the scutellum and coleorhiza cells, whereas the rest of the embryo tissues only had nuclei with a 2C and 4C DNA content. This study was accompanied by a spatiotemporal profile analysis of the DNA synthetic activity in the organs of Brachypodium embryos during germination using EdU labelling. Upon imbibition, nuclear DNA replication was initiated in the radicle within 11 h and subsequently spread towards the plumule. The first EdU-labelled prophases were observed after 14 h of imbibition. Analysis of selected genes that are involved in the regulation of the cell cycle, such as those encoding cyclin-dependent kinases and cyclins, demonstrated an increase in their expression profiles.

High-frequency clonal propagation of Curcuma angustifolia ensuring genetic fidelity of micropropagated plants

A high-frequency clonal propagation protocol was developed for Curcuma angustifolia Roxb., a high valued traditional medicinal plant. Axillary bud explants of C. angustifolia were explanted on Murashige and Skoog (MS) medium fortified with 4.4–22.2 µM 6-benzyladenine (BA), 2.9–5.7 µM indole-3-acetic acid (IAA), 2.3–23.2 µM kinetin (Kin), 2.7–5.4 µM naphthalene acetic acid (NAA) and 67.8-271.5 µM adenine sulphate (Ads) in different combinations. The maximum number of shoots per explants (14.1 ± 0.55) and roots per shoot (7.6 ± 0.47) was achieved on media containing 13.3 µM BA, 5.7 µM IAA and 135.7 µM Ads. Stability in phytomedicinal yield potential of micropropagated plants was assessed through GC–MS and HPTLC. Gas chromatogram of essential oil of conventional and micropropagated plants of C. angustifolia had similar essential oil profile. HPTLC analysis of rhizome extracts of in vitro and field grown plants revealed no significant differences in the fingerprint pattern and in curcumin content. Genetic integrity of in vitro and field grown derived plants were evaluated with inter-simple sequence repeat (ISSR) primers and flow cytometry using Glycine max as an internal standard. A total of 1260 well resolved bands were generated by 12 ISSR primers showing monomorphic banding patterns across all plants analyzed. The mean 2C DNA content of conventionally and micropropagated plant was estimated to be 2.26 pg and 2.31 pg, respectively. As no somaclonal variations were detected in tissue culture plantlets, the present micropropagation protocol could be applied for in vitro conservation and large-scale production of C. angustifolia.

Estimation of Nuclear DNA Content and Determination of Relationship Between Altitude and Genome Size of USDA Turkish Oat (Avena spp.) Collection

Crop improvement is an important approach to overcome challenges raised from future uncertainties of agricultural systems and growing human population. The fundamental need for such improvement efforts is the availability of well characterized plant germplasm with sufficient genetic diversity. The 2C DNA is defined as the nuclear DNA content of an unreplicated diploid cell (in G1 phase) and is used both to get an estimate of genome size and ploidy level. Flow cytometry provides accurate and fast estimation of the genome size of plants. The genus Avena belongs to Poaceae (Gramineae) family and includes approximately 30 species including common oat (A. sativa). There are 837 Avena accessions in the USDA (United States Department of Agriculture) Germplasm Resources Information Network (USDA-GRIN) collected from Turkey. This also is the largest ex situ Avena collection from Turkey. However, initial genomic characterization of the collection has not yet been conducted. We estimated genome sizes and determined ploidy levels of Turkish oat collection. Nuclear DNA content of accessions ranged from 25.66 to 26.56 pg for A. sativa, from 25.48 to 26.88 pg for A. sterilis and from 24.85 to 26.41 pg for A. fatua. The average and range values for all three hexaploid species were in a similar close range. The range for putative tetraploid accessions belonging to A. barbata was from 12.79 to 16.90 pg. We found a number of aggregates. Furthermore, there was a negative correlation found between altitude and genome size. Obtained results will help to better utilize Avena collection in breeding efforts.

Estimation of nuclear DNA content and its variation among Indian Tea accessions by flow cytometry.

Nuclear DNA content and genome size variation among 36 Indian tea accessions were analyzed by flow cytometry. Initial standardization of protocols for isolation of nuclei, DNA staining and selection of an internal standard for tea accessions which have significantly high amount of phenolic secondary metabolites in their cytosol was carried out. Results obtained revealed that 2C DNA content of Indian tea is 7.46pg which correspondsto 1C genome size of 3673Mb. Inter accession variation in 2C DNA content was also observed among 35 diploid taxa ranging from 7.23 to 7.73pg which was significant at 1% probability level. The 2C DNA content of triploid (UPASI 3) was observed to be 11.47pg which is concurrent with the expected value. Results obtained showed that Assam and Cambod type tea accession have higher 2C DNA content of 7.73pg whereas Assam Cambod hybrids and Assam China hybrids have reduction in DNA content with 2C amounts, 7.23 and 7.32pg DNA respectively. The present study suggests that the species involved in origin of Indian tea must have differed in their genome sizes owing to significant inter accession variation in nuclear DNA content.

Assessment of Iranian endemic Artemisia khorassanica: karyological, genome size, and gene expressions involved in artemisinin production.

The species of Artemisia, one of the largest genera of the family Asteraceae, are frequently utilized for the treatment of diseases such as malaria, hepatitis, cancer, inflammation, and infections by fungi, bacteria, and viruses. Karyological studies were performed on 18 Artemisia khorassanica populations: eleven were diploid (2n = 18) and seven were tetraploid (2n = 36). The mean chromosome lengths were 3.61 and 3.84 µm for diploids and tetraploids, respectively. Two chromosome types ("m", "sm") formed karyotype formulas "18m" for diploids and "36m" and "34m + 2sm" for tetraploids. The mean 2C DNA contents were 5.91 and 11.53 pg in diploids and tetraploids, respectively. The transcription levels of key genes involved in artemisinin production were compared in diploid (B, D, H) and tetraploid (O, P, R) A. khorassanica relative to A. annua as a standard species. No artemisinin content was detected in diploid and tetraploid A. khorassanica populations. No significant diefrences were detected between diploids and tetraploids in terms of DXR , HMGR, FDS, and ADS gene expression. This implies that most of the genomic amplification likely occurs in the amount of repetitive DNA and not in unique sequences. The DBR2 gene was expressed in the diploid A. khorassanica in a low amount but silenced in the autotetraploid A. khorassanica.

Primula veris plants derived from in vitro cultures and from seeds: genetic stability, morphology, and seed characteristics

Flow cytometry measurement confirmed the genetic stability of Primula veris L. (cowslip) plants obtained during the micropropagation process. The mean 2C DNA content was 0.97 pg/2C and P. veris can be classified in the group of plants of very small genomes. Comparative morphological analysis of cowslip plants regenerated in vitro and derived from seeds was carried out in three subsequent years under common garden conditions, which limited the effects of environmental factors. The origin of specimens significantly influenced most of the morphological traits examined. Plants that originated from seeds were characterized by more intensive development rate and higher total seed production compared to micropropagated plants. Significant differences between the two groups of specimens were observed for the number of flower stalks, flowers, fruits, and seeds per plant. No interseasonal variation within the reproductive traits such as number of seeds per flower stalk and per plant was found for plants derived from in vitro cultures. That allows prediction of seed production from in vitro regenerated plants after their successful acclimatization to field conditions. Importantly, plants derived from in vitro cultures set significantly bigger seeds. Our results are promising for broad uses of the regenerated in vitro cowslip plants in ornamental horticulture and the pharmaceutical industry.

DNA Replication during Seed Germination, Deterioration, and Its Relation to Vigor in Alfalfa and White Clover

Differences in seed vigor that arise due to seed aging, even in seeds with high levels of germination, can result in differences in field emergence. Current test methods to identify vigor differences in commercial seed lots with high standard germination take several days to complete. DNA replication, measured using flow cytometry as the ratio of 4C/2C DNA, during early germination of unaged and aged seeds of alfalfa (Medicago sativa L.) and white clover (Trifolium repens L.) was examined as a potential rapid and accurate method of assessing seed vigor. The relationship between 4C/2C DNA during germination of 10 commercial seed lots of both species and their field emergence was also determined. Most of the nuclei in dry mature seeds were arrested in the 2C DNA stage, with only a small proportion of 4C DNA nuclei. The proportion of 4C nuclei increased during germination of seeds, although the increase was lower and delayed in aged seeds with slower and lower germination. The 4C/2C ratio after 24‐h imbibition predicted the field emergence of all seed lots, but particularly of the lots with high standard germination (SG) (SG ≥ 75%, alfalfa, R2 = 0.815; SG ≥ 80%, white clover, R2 = 0.771; P < 0.01). Thus, DNA replication marks an advancement of germination in alfalfa and white clover seed, and the 4C/2C DNA ratio after 24‐h imbibition has potential to predict the seed vigor of both species.

Cytogenetics and Genome Size Evolution in Illicium L.

Illicium is an ancient genus and member of the earliest diverging angiosperms known as the Amborellales, Nymphaeales, and Austrobaileyales (ANA) grade. These adaptable, broadleaf evergreen shrubs, including ≈40 species distributed throughout Asia and North America, are valued for diverse culinary, medicinal, and ornamental applications. The study of cytogenetics of Illicium can clarify various discrepancies and further elucidate chromosome numbers, ploidy, and chromosome and genome size evolution in this basal angiosperm lineage and provide basic information to guide plant breeding and improvement programs. The objectives of this study were to use flow cytometry and traditional cytology to determine chromosome numbers, ploidy levels, and relative genome sizes of cultivated Illicium. Of the 29 taxa sampled, including ≈11 species and one hybrid, 2C DNA contents ranged from 24.5 pg for Illicium lanceolatum to 27.9 pg for Illicium aff. majus. The genome sizes of Illicium species are considerably higher than other ANA grade lineages indicating that Illicium went through considerable genome expansion compared with sister lineages. The New World sect. Cymbostemon had a slightly lower mean 2C genome size of 25.1 pg compared with the Old World sect. Illicium at 25.9 pg, providing further support for recognizing these taxonomic sections. All taxa appeared to be diploid and 2n = 2x = 28, except for Illicium floridanum and Illicium mexicanum which were found to be 2n = 2x = 26, most likely resulting from dysploid reduction after divergence into North America. The base chromosome number of x = 14 for most Illicium species suggests that Illicium are ancient paleotetraploids that underwent a whole genome duplication derived from an ancestral base of x = 7. Information on cytogenetics, coupled with phylogenetic analyses, identifies some limitations, but also considerable potential for the development of plant breeding and improvement programs with this genus.

DNA content estimation of Fig and Black Mulberry using flow cytometry

Abstract In this study, fig and black mulberry DNA contents were estimated using DAPI fluorescence stain in flow cytometry. The 2C DNA contents of the fig and black mulberry were found as 0.82 pg and 8.34 pg, respectively. The calculated 1C value of genome size of fig is 401.8 Mbp and that of black mulberry is 4086.6 Mbp. The ratio of 2C DNA content and 1C genome of the black mulberry was 10.17 times that of the fig although fig is diploid and black mulberry is decosaploid.

Molecular Reconstruction of an Old Pedigree of Diploid and Triploid Hydrangea macrophylla Genotypes.

The ornamental crop species Hydrangea macrophylla exhibits diploid and triploid levels of ploidy and develops lacecap (wild type) or mophead inflorescences. In order to characterize a H. macrophylla germplasm collection, we determined the inflorescence type and the 2C DNA content of 120 plants representing 43 cultivars. We identified 78 putative diploid and 39 putative triploid plants by flow cytometry. In our collection 69 out of 98 flowering plants produced lacecap inflorescences, whereas 29 plants developed mophead inflorescences. Surprisingly, 12 cultivars included diploid as well as triploid plants, while 5 cultivars contained plants with different inflorescence types. We genotyped this germplasm collection using 12 SSR markers that detected 2–7 alleles per marker, and identified 51 different alleles in this collection. We detected 62 distinct fingerprints, revealing a higher genetic variation than the number of cultivars suggested. Only one genotype per cultivar is expected due to the vegetative propagation of Hydrangea cultivars; however we identified 25 cultivars containing 2–4 different genotypes. These different genotypes explained the variation in DNA content and inflorescence type. Diploid and triploid plants with the same cultivar name were exclusively mix-ups. We therefor assume, that 36% of the tested plants were mislabeled. Based on the “Wädenswil” pedigree, which includes 31 of the tested cultivars, we predicted cultivar-specific fingerprints and identified at least 21 out of 31 cultivars by SSR marker-based reconstruction of the “Wädenswil” pedigree. Furthermore, we detected 4 putative interploid crosses between diploid and triploid plants in this pedigree. These interploid crosses resulted in diploid or/and triploid offspring, suggesting that crosses with triploids were successfully applied in breeding of H. macrophylla.

Karyotype diversity and 2C DNA content in species of the Caesalpinia group

BackgroundThe Leguminosae family is the third-largest family of angiosperms, and Caesalpinioideae is its second-largest subfamily. A great number of species (approximately 205) are found in the Caesalpinia group within this subfamily; together with these species’ phenotypic plasticity and the similarities in their morphological descriptors, make this a complex group for taxonomic and phylogenetic studies. The objective of the present work was to evaluate the karyotypic diversity and the 2C DNA content variation in 10 species of the Caesalpinia group, representing six genera: Paubrasilia, Caesalpinia, Cenostigma, Poincianella, Erythrostemon and Libidibia. The GC-rich heterochromatin and 45S rDNA sites (which are used as chromosome markers) were located to evaluate the karyotype diversity in the clade. The variation in the 2C DNA content was determined through flow cytometry.ResultsThe fluorochrome banding indicated that the chromomycin A3+/4′,6-diamidino-2-phenylindole− blocks were exclusively in the terminal regions of the chromosomes, coinciding with 45S rDNA sites in all analyzed species. Physical mapping of the species (through fluorescence in situ hybridization) revealed variation in the size of the hybridization signals and in the number and distribution of the 45S rDNA sites. All hybridization sites were in the terminal regions of the chromosomes. In addition, all species had a hybridization site in the fourth chromosome pair. The 2C DNA content ranged from 1.54 pg in Erythrostemon calycina to 2.82 pg in the Paubrasilia echinata large-leaf variant. The Pa. echinata small-leaf variant was isolated from the other leaf variants through Scoot-Knott clustering.ConclusionsThe chromosome diversity and the variation in the 2C DNA content reinforce that the actual taxonomy and clustering of the analyzed taxa requires more genera that were previously proposed. This fact indicates that taxonomy, phylogeny and cytoevolutionary inference related to the complex Caesalpinia group have to be done through integrative evaluation.

Flow Sitometri ile Bazı Ispanak Aksesyonlarının Çekirdek DNA İçeriklerinin Belirlenmesi

The aim of this research is to determine ploidy levels of 53 spinach (Spinacia oleracea L.) accessions obtained from abroad sources by using flow cytometry. According to the results of the statistical analysis, the differences among DNA content of accessions were statistically important. The average DNA content of spinach accessions used in the study ranged between 2.225 pg/2C (Esfenaj accession) and 2.059 pg/2C (Matador and Godir accessions). The mean nuclear DNA of accessions shown to be relatively stable due to low standard deviation (0.003-0.096). Based on the mitotic chromosome analyses of the some plants with different DNA contents, mitotic chromosome number of the all plants analyzed in the study were determined as 2n=12. These results indicate that all the accessions used in the study are diploid although their DNA content is significantly vary. According to the clustering analysis of the 2C DNA contents results, it was observed that eight sub clusters were formed under two main clusters. The clustering of axioms was performed in a similar manner to the multiple comparison test. As a result, the data will save time and labor convenience in a further breeding studies deal with the same 53 spinach accessions of our study.

Data reassessment in a phylogenetic context gives insight into chromosome evolution in the giant genus Solanum (Solanaceae)

Chromosome data are fundamental in evolution. However, there has been no attempt to synthesize and evaluate the significance of such information from a phylogenetic perspective in the giant genus Solanum, which was the aim of this work. New and published information of the main cytotaxonomic features (chromosome number, polyploidy, total length of the haploid complement, mean chromosome length, mean arm ratio, karyotype formula, nuclear DNA amount, number/position of rDNA sites) was compiled and mapped onto an embracing Solanaceae phylogeny, performing Ancestral States Reconstruction. There were 506 Solanum species with chromosome counts (49.7% from an estimated total of 1,018 spp.), with x = 12 being the most frequent number (97%). Species with karyotypes represent 18.8%, while 8% have been studied with any molecular cytogenetic technique. Chromosome characters showed transitions associated with supported nodes, some of which have undergone fewer transitions than others. The common ancestor of all Solanum was a diploid with 2n = 24, a karyotype with st and/or t chromosomes, 2C DNA content of 1–1.2 pg, one locus of 18–5.8–26S rDNA and one of 5S, both loci being asyntenic. The chromosomal variables behave as homoplastic, with reversions in all branches. The analysed characters were sorted from more to less conserved: asynteny of rDNA loci; number of sites of 18–5.8–26S; chromosome number; karyotype formula; number of 5S loci. This pattern of chromosomal evolution distinguishes Solanum from closely related genera and from genera from other families with a similar number of species.

Karyology of Agapanthus africanus (L.) Hoffmanns (Agapanthaceae)

The genus Agapanthus L'Héritier (Agapanthaceae) is a monocotyledonous, herbaceus and perennial plant group endemic to southern Africa. Ten species have been described for this group, in which three are considered evergreen and seven deciduous plants. Nevertheless, Agapanthus L'Héritier taxonomy is considered very difficult once there are few unique characters and a high plasticity depending on circumstances of growing. This feature takes some authors to believe in the existence of only one species with innumerous varieties.The cytogenetic has been an important tool to aid the plant taxon identification, and to date, all taxa of Agapanthus L'Héritier studied cytologically presented 2n = 30. Although the species possess large chromosomes, few karyological studies has been explored. This work aimed to increase the cytogenetic knowledge of Agapanthus africanus (L.) Hoffmanns by chromosome banding techniques with DAPI / CMA3 and Fluorescent in situ Hybridization (FISH). In addition, flow cytometry was used to determinate DNA content and the percentage of AT / GC nitrogenous bases. Plants studied showed 2n = 30 chromosomes, ranging from 4.34 - 8.55 µm, with karyotype formulae (KF) = 10m + 5sm. The FISH technique revealed one 45S rDNA signal observed proximally to centromere of the chromosome 7, and one 5S rDNA signal observed proximally to centromere of chromosome 9. The 2C DNA content estimated for the species was 2C = 24.4pg, with 59% of AT and 41% of GC. Our data allowed important upgrade for biology and cytotaxonomy of Agapanthus africanus (L.) Hoffmanns, and will be used to future taxonomic and phylogenetics studies, as well as for the chromosome evolution of the genus. APOIO FINANCEIRO: Fapemig, Capes, CNPq

Growth, development and steviol glycosides content in the relation to the photosynthetic activity of several Stevia rebaudiana Bertoni strains cultivated under temperate climate conditions

Abstract The objective of this study was to evaluate the development and the quality of leaves from several S. rebaudiana Bertoni strains cultivated under temperate climate conditions in the tunnel and in the open field in the relation to the photosynthetic activity. Investigated stevia strains differed significantly in terms of whole plant and leaf morphology, photosynthetic efficiency as well as in the content of steviol glycosides, and antioxidant capacity, independently of cultivation place. On the basis of several chlorophyll a fluorescence parameters analysis it was stated that plants of all examined strains, did not exhibit photosynthesis inhibition. The studies of the relationship between photosynthetic activity and the level of secondary metabolites indicated that the highest content of steviol glycosides and phenolic compounds had plants showing also the highest value of the energy fraction photochemically converted in PS II (YII), while the highest ratio of rebaudioside A to stevioside content corresponded with low amount of energy dissipated as a heat (YNPQ). This results might indicate that steviol glycosides play a role in the better adaptation to environmental conditions. Karyology and cytometric studies revealed that all investigated stevia strains were diploids without significant differences in 2C DNA amount. Therefore, observed diversity in the content of steviol glycosides and phenolic compounds between investigated S. rebaudiana strains was not the effect of polyploidization but probably resulted from the differences in the metabolic activity of the examined plants.

Genome size and ploidy level among wild and cultivated Prunus taxa in Slovakia

2C DNA content and ploidy level variation of Prunus spinosa and closely related taxa together with Prunus domestica L. and Prunus insititia L. was studied in Slovakia. The aim of the study was to define genome sizes and find differences between closely related taxa within Prunus spinosa sensu lato mentioned in previous works. According to our results, investigated taxa can be divided into three groups according to ploidy level: Prunus spinosa, Prunus dasyphylla, Prunus ×fruticans, Prunus ×dominii and Prunus ×schurii are tetraploids, Prunus ×fechtneri is pentaploid, and P. domestica and P. insititia are hexaploids. Genome size differences within tetraploid taxa were relatively small (Prunus spinosa: 1.40 ± 0.02, P. ×domini: 1.44 ± 0.01, P. ×fruticans: 1.48 ± 0.02, P. ×schurii: 1.44 ± 0.02), but statistically significant. Although further research is needed, it seems that the concept of several taxa as product of hybridization between P. spinosa and cultivated plum species has been supported by our study.

Variation in nuclear DNA content and chromosome numbers in blueberry

Abstract Commercial blueberry production in the U.S. relies on cultivars derived from combinations of different blueberry species. Interspecific hybridization continues to be a vital strategy for blueberry breeding, especially in regards to improving abiotic and biotic stress tolerance to expand the range of production. Blueberry species have many different genome sizes, presumably due to differing ploidy levels and/or DNA content. In some species, such as Vaccinium corymbosum L. and V. pallidum Aiton, there are also different ploidy series available for breeding, further complicating the observed DNA content in elite selections. In this study we determined DNA content in 77 blueberry taxa including 11 species and 64 hybrid cultivars. Among these taxa are 11 diploid, 49 tetraploid, one pentaploid, and 16 hexaploid plants representing southern highbush, northern highbush, low bush, rabitteye, and half high blueberries. DNA content was measured for three separate tissue samples from at least three individual plants per taxa for accuracy. Absolute genome sizes varied significantly (P = 0.05) among ploidy levels, with 2C-value means of 1.09 pg for diploid, 2.10 pg for tetraploid, and 3.60 pg for hexaploid taxa. Holoploid genome size (2C DNA) was significantly correlated with ploidy or presumed chromosome number (2n) (r = 0.96, P = 0.0001) but only weakly correlated with monoploid genome size (1Cx-DNA) (r = 0.43, P = 0.0001).

Cell cycle in egg cell and its progression during zygotic development in rice.

Rice egg is arrested at G1 phase probably by OsKRP2. After fusion with sperm, karyogamy, OsWEE1-mediated parental DNA integrity in zygote nucleus, zygote progresses cell cycle to produce two-celled embryo. In angiosperms, female and male gametes exist in gametophytes after the complementation of meiosis and the progression of nuclear/cell division of the haploid cell. Within the embryo sac, the egg cell is specially differentiated for fertilization and subsequent embryogenesis, and cellular programs for embryonic development, such as restarting the cell cycle and de novo gene expression, are halted. There is only limited knowledge about how the cell cycle in egg cells restarts toward zygotic division, although the conversion of the cell cycle from a quiescent and arrested state to an active state is the most evident transition of cell status from egg cell to zygote. This is partly due to the difficulty in direct access and analysis of egg cells, zygotes and early embryos, which are deeply embedded in ovaries. In this study, precise relative DNA amounts in the nuclei of egg cells, developing zygotes and cells of early embryos were measured, and the cell cycle of a rice egg cell was estimated as the G1 phase with a 1C DNA level. In addition, increases in DNA content in zygote nuclei via karyogamy and DNA replication were also detectable according to progression of the cell cycle. In addition, expression profiles for cell cycle-related genes in egg cells and zygotes were also addressed, and it was suggested that OsKRP2 and OsWEE1 function in the inhibition of cell cycle progression in egg cells and in checkpoint of parental DNA integrity in zygote nucleus, respectively.

Insights into nuclear DNA content, hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid.

Malaxis wallichii (Lindl.) Deb, a small, perennial, monopodial, terrestrial orchid, is endemic to tropical Himalayas at an altitude of 1200-2000m asl. The pseudobulbs are important ingredients of century old drug 'Ashtavarga' and a polyherbal energetic tonic 'Chyavanprash'. An efficient genetically stable in vitro propagation protocol using transverse thin cell layer culture system was established for M. wallichii. In the present report, meta-topolin alone proved to be three times more beneficial compared to other routinely used cytokinins in inducing highest number of shoot buds, plant height and growth of regenerated shoots. The highest regeneration frequency (89%) along with maximum number of adventitious shoots per explant (22.5±0.6) was observed in MS medium supplemented with 1.0mg/l meta-topolin and 0.5mg/l α-naphthalene acetic acid. Highest rooting frequency with highest number of roots (8.66±0.3) was achieved in half-strength MS medium fortified with 1.0mg/l indole acetic acid. Clonal stability of in vitro-derived plantlets was evaluated and compared to donor plant using intron splice junction (ISJ) markers and flow cytometry. ISJ markers revealed 4.76% clonal variability indicating high degree of genetic stability amongst the in vitro-derived regenerants. The nuclear DNA content of M. wallichii (2n) was found to be 2C=2.760±0.02pg and therefore, 1349.64Mbp (1C). Flow cytometry analysis of actively growing young and mature leaves from donor as well as in vitro-derived plantlets revealed presence of three peaks corresponding to 2C, 4C and 8C, while 2C was the most abundant. In the present investigation, there was no significant difference in the 2C DNA content between the mother and in vitro-derived plants; however, the frequency of endopolyploid cells varied in young and adult plants. An increased H2O2 content as well as lipid peroxidation activities were observed during early stages of acclimatization which declined afterwards. The enhanced activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase in acclimatized plantlets as compared to in vitro-grown ones revealed their active involvement in growth and development against oxidative stress under external adverse environment.

Visualization of chromosome condensation in plants with large chromosomes

BackgroundMost data concerning chromosome organization have been acquired from studies of a small number of model organisms, the majority of which are mammals. In plants with large genomes, the chromosomes are significantly larger than the animal chromosomes that have been studied to date, and it is possible that chromosome condensation in such plants was modified during evolution. Here, we analyzed chromosome condensation and decondensation processes in order to find structural mechanisms that allowed for an increase in chromosome size.ResultsWe found that anaphase and telophase chromosomes of plants with large chromosomes (average 2C DNA content exceeded 0.8 pg per chromosome) contained chromatin-free cavities in their axial regions in contrast to well-characterized animal chromosomes, which have high chromatin density in the axial regions. Similar to animal chromosomes, two intermediates of chromatin folding were visible inside condensing (during prophase) and decondensing (during telophase) chromosomes of Nigella damascena: approximately 150 nm chromonemata and approximately 300 nm fibers. The spatial folding of the latter fibers occurs in a fundamentally different way than in animal chromosomes, which leads to the formation of chromosomes with axial chromatin-free cavities.ConclusionDifferent compaction topology, but not the number of compaction levels, allowed for the evolution of increased chromosome size in plants.