2C DNA Content Research Articles

Direct somatic embryogenesis and flow cytometric assessment of ploidy stability in regenerants of Caladium × hortulanum ‘Fancy’

Caladium × hortulanum 'Fancy' is an important ornamental plant grown in pots and landscapes and known for its colorful leaves often used for interior decorations. In this work, we present a method of in vitro regeneration from three explants source through direct somatic embryogenesis (DSE) wherein the regenerated plants were screened for ploidy changes through flow cytometry analysis. Tuber, leaf and petiole explants were cultured on MS basal medium supplemented with 1-napthalene acetic acid (NAA), 6-benzyl amino purine (BAP) and N-phenyl-N'-1, 2,3-thiadiazol-5-ylurea (TDZ) concentrations. Tuber explants induced highest direct somatic embryos on NAA (1 mg L- 1) + BAP (0.5 mg L- 1) with 55.6 mean number of embryos per explant while as leaf and petiole explants amended with 1 mg L- 1 TDZ developed 18.7 and 12.27 mean number of embryos per explant respectively. The highest embryo conversion frequency was achieved on BAP (2 mg L- 1) + NAA (0.2 mg L- 1) with 44.2, 18.7 and 7.5 mean number of plantlets produced per tuber, leaf and petiole explant respectively after 4 weeks of culture. Plantlets were later rooted and maximum number of roots (6.33) per shoot was achieved on 2 mg L- 1 indolebutyric acid amended medium. Description of the process of DSE is presented through the histological and SEM evidences. The 2C DNA content of field grown plants and the DSE regenerants evaluated under flow cytometric analysis were 8.06 pg and 8.28 pg respectively showing no ploidy changes. Hence, a successful protocol of inducing direct somatic embryos from three explant types with efficient embryo conversion frequency was obtained with regenerants showing similar DNA ploidy as that of their parent plants.

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Genome Size, Chromosome Number and Morphological Data Reveal Unexpected Infraspecific Variability in Festuca (Poaceae)

Polyploidy has played an important evolutionary role in the genus Festuca (Poaceae), and several ploidy levels (ranging from 2n = 2x = 14 to 2n = 12x = 84) have been detected to date. This study aimed to estimate the genome size and ploidy level of two subspecies belonging to the F. yvesii polyploid complex by flow cytometry and chromosome counting. The phenotypic variation of the cytotypes was also explored, based on herbarium material. The genome size of F. yvesii subsp. lagascae has been estimated for the first time. Nuclear 2C DNA content of F. yvesii subsp. summilusitana ranged from 21.44 to 31.91 pg, while that of F. yvesii subsp. lagascae was from 13.60 to 22.31 pg. We report the highest ploidy level detected for Festuca (2n = 14x = 98) and previously unknown cytotypes. A positive correlation between holoploid genome size and chromosome number counts shown herein was confirmed. The morphometric approach showed a slight trend towards an increase in the size of some organs consistent with the variation in the ploidy level. Differences in characters were usually significant only among the most extreme cytotypes of each subspecies, but, even in this case, the high overlapping ranges prevent their distinction.

Ploidy differentiation and floral scent divergence in Buddleja macrostachya (Scrophulariaceae) complex

Floral scent has been suggested to be a key factor both in the diversification of the angiosperms, and the evolution and intra-specific differentiation of plants. In this study, we determined the ploidy and nuclear DNA content of B. macrostachya plants from 14 different populations across Yunnan using flow cytometry. The components of the floral scent were also identified using gas chromatography and mass spectrometry (GC-MS). Within these 14 populations, two new ploidy levels, tetraploid (2n = 4x) and octaploid (2n = 8x) were detected and identified for the first time here. The mean 2C DNA content of the tetraploids ranged from 1.81 ± 0.01 to 1.92 ± 0.02 G, but was 3.52 ± 0.15 to 3.73 ± 0.04 G in the octaploids. In addition, thirty-two scent compounds were identified, most of which could be categorized into four major classes: fatty acid derivatives (49.60 ± 7.45%); benzenoids (21.24 ± 3.96%); monoterpenes (25.28 ± 3.70%) and sesquiterpenes (3.88 ± 1.05%). Both tetraploids and octaploids emitted large relative amounts of 4-hydroxy-4-methyl-2-pentanone (42.21 ± 3.52% and 21.57 ± 3.10%, respectively) and benzadehyde (20.42 ± 2.35% and 28.45 ± 2.37%, respectively). Multivariate analysis of floral scent chemistry revealed marked dissimilarity (49.49%) between tetraploids and octaploids, and 4-hydroxy-4-methyl-2-pentanone and benzadehyde contributed greatly to this divergence in chemical scent profile. Our results suggest that B. macrostachya is a species complex with four different ploidy levels (2n = 4x, 6x, 8x, 12x). We believe the complex is in a process of pollinator-mediated rapid divergence.

First report on genome size and ploidy determination of five indigenous coffee species using flow cytometry and stomatal analysis

The nuclear 2C DNA content and ploidy level determination of five indigenous coffee species from India was carried out using flow cytometry and by assessing the stomatal characteristics. The nuclear DNA content (2C/pg) analyzed varied from 1.29 pg in Coffea wightiana Wall. ex Wight & Arn. to 1.48 pg in Coffea jenkinsii Hook.f. Based on the genome size, all five species were divided into two groups. The first group comprises Coffea travancorensis Wight & Arn and C. wightiana with lower genome size (2C DNA 1.29–1.30 pg), and the second group consists of Coffea bengalensis Roxb.ex Schult, Coffea khasiana (Korth.) Hook.f and C. jenkinsii with larger genome size (2C DNA 1.45–1.48 pg). The species included in the first group were inhabited in dry environmental conditions in contrast to the species included in the second group which was predominantly from mesic habitat. Among the stomatal characteristics, no significant variation in stomatal density was observed among the species, although stomatal guard cell length and stomatal chloroplast number recorded significant variation among some species. Based on flow cytometry and stomatal characteristics, all five endangered coffee species analyzed in the study were identified as diploids. The study revealed that stomatal guard cell length and stomatal chloroplast number could be fast, inexpensive, and reliable methods for determining the ploidy status of indigenous coffee species.

Planarian stem cells specify fate yet retain potency during the cell cycle.

Planarian whole-body regeneration is enabled by stem cells called neoblasts. At least some neoblasts are individually pluripotent. Neoblasts are also heterogeneous, with subpopulations of specialized neoblasts having different specified fates. Fate specification in neoblasts is regulated by fate-specific transcription factor (FSTF) expression. Here, we find that FSTF expression is common in neoblast S/G2/M cell-cycle phases but less common in G1. We find that specialized neoblasts can divide to produce progeny with asymmetric cell fates, suggesting that they could retain pluripotency. Furthermore, no known neoblast class was present in all neoblast colonies, suggesting that pluripotency is not the exclusive property of any known class. We tested this possibility with single-cell transplantations, which indicate that at least some specialized neoblasts are likely clonogenic. On the basis of these findings, we propose a model for neoblast pluripotency in which neoblasts can undergo specialization during the cell cycle without loss of potency.

Genome Size Diversity in Rare, Endangered, and Protected Orchids in Poland.

Orchidaceae is one of the largest and the most widespread plant families with many species threatened with extinction. However, only about 1.5% of orchids’ genome sizes have been known so far. The aim of this study was to estimate the genome size of 15 species and one infraspecific taxon of endangered and protected orchids growing wild in Poland to assess their variability and develop additional criterion useful in orchid species identification and characterization. Flow cytometric genome size estimation revealed that investigated orchid species possessed intermediate, large, and very large genomes. The smallest 2C DNA content possessed Liparis loeselii (14.15 pg), while the largest Cypripedium calceolus (82.10 pg). It was confirmed that the genome size is characteristic to the subfamily. Additionally, for four species Epipactis albensis, Ophrys insectifera, Orchis mascula, Orchis militaris and one infraspecific taxon, Epipactis purpurata f. chlorophylla the 2C DNA content has been estimated for the first time. Genome size estimation by flow cytometry proved to be a useful auxiliary method for quick orchid species identification and characterization.

Developing partial interspecific hybrids of Momordica charantia × Momordica balsamina and their advance generations

Interspecific hybridization and introgression breeding are the major approaches employed to address climate change and food security challenges globally. The present study was conducted to trace the nature of crossing barriers and to introgress novel genes from Momordica balsamina to cultivated bitter gourd Momordica charantia. The hybridization success rate was very low between these two species with a higher rate of seedling mortality. A total of 116 accessions of Momordica charantia (female parent) were used for artificial cross-pollination with Momordica balsamina (IC-467683). Among these, only six female parents belonging to Momordica charantia (DBGS-2, NEH-4, PDMGY-201, Pusa Aushadhi, DBGS-3, and NEH-3) exhibited variable percent fruit set when crossed with Momordica balsamina. Among these interspecific crosses, only one cross combination (Pusa Aushadhi × Momordica balsamina) produced the higher percentage of normal seeds and only one seed was germinated followed by development into a full plant. The pollen-pistil interaction study confirmed the existence of post dynamic barriers in all these unsuccessful crosses. A single plant progeny of M. charantia × M. balsamina was derived and its advanced generations were studied at morphological, cytological, and molecular levels. The plant phenotype predominately resembled the female parent with variation in a few traits. The cytological analysis depicted the normal meiosis and the genomic 2C DNA content of the interspecific hybrid was higher than the female parent. The microsatellite (SSRs) markers analysis showed that amplified fragments of interspecific hybrids exhibited highly conserved similarity with fragments of female parents with a very low frequency of male fragments. The F2 and backcross generations were produced from the single partial interspecific hybrid (Pusa Aushadhi × M. balsamina). Twenty advanced generation F2 plants showed variation with majority traits resembles the female parent. The microsatellite analysis revealed that the progenies of backcross generation amplified the fragments with high similarity to both female (Pusa Aushadhi; M. charantia) and the male parent (M. balsamina). These results confirmed the partial introgression of chromosome segments of M. balsamina. But further generations could not be recovered and advanced generation was died because of chlorosis. It was the first attempt to develop an interspecific partial hybrid and its backcross population which would provide preliminary information on the crossability relationship between these two species for transferring novel gene/s or traits from M. balsamina to M. charantia.

Detection of ploidy and chromosomal aberrations in commercial oil palm using high-throughput SNP markers.

Karyotyping using high-density genome-wide SNP markers identified various chromosomal aberrations in oil palm (Elaeis guineensis Jacq.) with supporting evidence from the 2C DNA content measurements (determined using FCM) and chromosome counts. Oil palm produces a quarter of the world's total vegetable oil. In line with its global importance, an initiative to sequence the oil palm genome was carried out successfully, producing huge amounts ofsequence information, allowing SNP discovery. High-capacity SNP genotyping platforms have been widely used for marker-trait association studies in oil palm. Besides genotyping, a SNP array is also an attractive tool for understanding aberrations in chromosome inheritance. Exploiting this, the present study utilized chromosome-wide SNP allelic distributions to determine the ploidy composition of over 1,000 oil palms from a commercial F1 family, including 197 derived from twin-embryo seeds. Our method consisted of an inspection of the allelic intensity ratio using SNP markers. For palms with a shifted or abnormal distribution ratio, the SNP allelic frequencies were plotted along the pseudo-chromosomes. This method proved to be efficient in identifying whole genome duplication (triploids) and aneuploidy. We also detected several loss of heterozygosity regions which may indicate small chromosomal deletions and/or inheritance of identical by descent regions from both parents. The SNP analysis was validated by flow cytometry and chromosome counts. The triploids were all derived from twin-embryo seeds. This is the first report on the efficiency and reliability of SNP array data for karyotyping oil palm chromosomes, as an alternative to the conventional cytogenetic technique. Information on the ploidy composition and chromosomal structural variation can help to better understand the genetic makeup of samples and lead to a more robust interpretation of the genomic data in marker-trait association analyses.

Genetic and biochemical stability of in vitro raised and conventionally propagated Centella asiatica - A valuable medicinal herb

An efficient micropropagation protocol was developed for C. asiatica, a commercially important herb having high medicinal and cosmetic properties. Shoot induction was observed on Murashige and Skoog (MS) medium fortified with various combinations of benzyladenine (BA) (2.22 - 22.2 µM) and naphthalene acetic acid (NAA) (1.34 - 8.06 µM). The maximum number of shoots (11.30 ± 0.68) per explant was initiated on MS media containing BA (6.66 µM) and NAA (2.69 µM). Maximum shoot proliferation was achieved on MS medium supplemented with BA (6.66 µM) and indole-3-acetic acid (IAA) (2.84 µM). The best rooting (24.69 ± 0.08) per explant was achieved on half-strength MS medium supplemented with indole-3-butyric acid (IBA) (4.92 µM). The in vitro regenerated plantlets were acclimatized and shifted to the field. Genetic stability of the micropropagated regenerants was evaluated using inter-simple sequence repeat (ISSR) markers and flow cytometry. Out of 28 primers tested, 9 primers resulted in 561 distinct and reproducible bands ranging from 200 to 1400 bp. Monomorphic banding profile was observed between the mother plant and tissue culture regenerants. The mean 2C DNA content of mother and micropropagated plants evaluated by flow cytometry were estimated to be 0.14 and 0.12 pg, thereby indicating no significant change in the 2C DNA content. Additionally, HPLC (High performance liquid chromatography) analysis did not show any significant differences in the content of triterpenes and phenolic compounds in both mother and micropropagated plants. Thus, this protocol could be implemented for large scale cultivation of elite germplasm of C. asiatica.

Mass propagation through direct and indirect organogenesis in three species of genus Zephyranthes and ploidy assessment of regenerants through flow cytometry.

Genus Zephyranthes consists of economically important plant species due to their high ornamental value and presence of valuable bioactive compounds. However, this genus propagates by asexual division only which gives slow propagation rate. Plant tissue culture has the potential to provide efficient techniques for rapid multiplication and genetic improvement of the genus. In this work, a dual in vitro regeneration system through callus mediated shoot regeneration and direct shoot regeneration in species Zephyranthes candida, Zephyranthes grandiflora and Zephyranthes citrina was investigated. Bulb, leaf and root explants were cultured on Murashige and Skoog (MS) medium amended with different plant growth regulators (PGR's) viz. 2,4-dichlorophenoxyacetic acid (2,4-D), 1-Naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), N-phenyl-N'-1,2,3 -thiadiazol-5-ylurea (TDZ), 6-Furfuryl- aminopurine (KIN) alone or in combinations for callus induction and regeneration. Only bulb explants showed callus induction and regeneration response on different PGR combinations with a varied response in callus induction percentage, callus color and callus texture. Creamish compact callus (CC) was induced on 2 mg L[Formula: see text] 2,4-D, brown friable callus (BF) on 2 mg L[Formula: see text] NAA + 1 mg L[Formula: see text] BAP and green friable callus (GF) callus on 1 mg L[Formula: see text] KIN + 3 mg L[Formula: see text] NAA. The maximum shoot multiplication from different callus types (indirect organogenesis) was achieved on 2 mg L[Formula: see text] BAP alone without combinations. Bulb explants of Z. grandiflora induced maximum callus induction percentage (86.4%) and shoot regeneration percentage (83.5%) with the maximum 08 shoots per 150 mg callus mass. The induction and regeneration response was followed in the order of Z. grandiflora > Z. candida > Z. citrina. Similarly, maximum direct organogenesis from bulb explants was obtained in Z. grandiflora (93.3%) followed by Z. candida (91.5%) and Z. citrina (90.4%) on 3 mg L[Formula: see text] TDZ amended MS media. Adventitious root induction was achieved on 2 mg L[Formula: see text] IBA with a maximum of 8 roots per shoot. The in vitro raised plantlets were successfully acclimatized in the field with 85% survival efficiency. The genome size (2C DNA content) of the field-grown plants and in vitro regenerated plants, evaluated through flow cytometry technique, were similar and showed no ploidy changes. An efficient mass propagation protocol was established for obtaining plants with unaltered genome size in the three species of Zephyranthes.

Targeted generation of polyploids in Hydrangea macrophylla through cross-based breeding

BackgroundUp to now, diploid and triploid cultivars were reported for the ornamental crop Hydrangea macrophylla. Especially, the origin of triploids and their crossing behaviors are unknown, but the underlying mechanisms are highly relevant for breeding polyploids.ResultsBy screening a cultivar collection, we identified diploid, triploid, tetraploid and even aneuploid H. macrophylla varieties. The pollen viability of triploids and tetraploids was comparable to that of diploids. Systematic crosses with these cultivars resulted in viable diploid, triploid, tetraploid and aneuploid offspring. Interestingly, crosses between diploids produced diploid and 0 or 1–94% triploid offspring, depending on the cultivars used as pollen parent. This finding suggests that specific diploids form unreduced pollen, either at low or high frequencies. In contrast, crosses of triploids with diploids or tetraploids produced many viable aneuploids, whose 2C DNA contents ranged between the parental 2C values. As expected, crosses between diploid and tetraploid individuals generated triploid offspring. Putative tetraploid plants were obtained at low frequencies in crosses between diploids and in interploid crosses of triploids with either diploid or tetraploid plants. The analysis of offspring populations indicated the production of 1n = 2x gametes for tetraploid plants, whereas triploids produced obviously reduced, aneuploid gametes with chromosome numbers ranging between haploid and diploid level. While euploid offspring grew normally, aneuploid plants showed mostly an abnormal development and a huge phenotypic variation within offspring populations, most likely due to the variation in chromosome numbers. Subsequent crosses with putative diploid, triploid and aneuploid offspring plants from interploid crosses resulted in viable offspring and germination rates ranging from 21 to 100%.ConclusionsThe existence of diploids that form unreduced pollen and of tetraploids allows the targeted breeding of polyploid H. macrophylla. Different ploidy levels can be addressed by combining the appropriate crossing partners. In contrast to artificial polyploidization, cross-based polyploidization is easy, cheap and results in genetically variable offspring that allows the direct selection of more robust and stress tolerant polyploid varieties. Furthermore, the generation of polyploid H. macrophylla plants will favor interspecific breeding programs within the genus Hydrangea.

Glucose limitation and pka1 deletion rescue aberrant mitotic spindle formation induced by Mal3 overexpression in Schizosaccharomyces pombe.

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe. We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1-308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1-241) both resulted in more severe phenotypes than Mal3 (1-308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446, was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.

Application of Horticultural and Tissue Culture Methods for Ex Situ Conservation of Endangered Primula farinosa L.

Our study aimed at active conservation of the last location of <em>Primula farinosa</em>, an endangered species in Poland, and assessed reproduction by seeds and plant propagation on sterile media in tissue culture conditions. We identified gibberellic acid (GA<sub data-id="subscript-1">3</sub>) as the key factor stimulating germination of <em>P. farinosa</em> seeds. Growing juvenile plants under controlled temperature of 18/16 °C day/night yielded good quality plant material without mycorrhization. In tissue culture, the most favorable medium for shoot propagation was MS supplemented with the lowest tested concentration of indole-3-butyric acid (IBA; 0.05 mg dm<sup data-id="superscript-1">−3</sup>) and 6-benzyl-aminopurine (BAP; 0.1 mg dm<sup data-id="superscript-2">−3</sup>). The rooting ability of shoots was high and comparable for all auxins used. 2C DNA content of seed-derived and micropropagated plants did not indicate any change in the ploidy level during in vitro cultivation. Plants derived from seeds and tissue cultures were compared in a 2-year study. Of all the characteristics compared, only the number of flowers per inflorescence was lower for micropropagated plants when compared with the seed-origin plants in the first year of observation. The difference was of transient nature and was not observed in the second year of the study. Effective protocols for in vivo and in vitro propagation of <em>P. farinosa</em> were developed, which can be used in practical species protection.

Cytogenetic and pollen identification of genus Gagnepainia (Zingiberaceae) in Thailand.

Gagnepainia godefroyi K. Schumann, 1904 and G. harmandii K. Schumann, 1904 belong to the genus Gagnepainia K. Schumann, 1904 of the Ginger family. They have the potential to be developed as medicinal and attractive ornamental plants. To date, the knowledge on the cytological and reproductive aspects of Gagnepainia have not been publicly available. Therefore, the aims of this research are to investigate the cytogenetic and pollen characters of Gagnepainia species using light, fluorescence, and scanning electron microscopes. The regular meiotic figures of 15 bivalents are found in both species and presented for the first time. These evidences indicate that Gagnepainia is diploid and contains 2n = 2x = 30 with basic number of x = 15. The mean nuclear DNA contents range from 1.986 pg in Gagnepainia sp., 2.090 pg in G. godefroyi to 2.195 pg in G. harmandii. Pollens of all species are monad, inaperturate, prolate with bilateral symmetry, and thick wall with fossulate exine sculpturing. The pollen size of G. harmandii (74.506 ± 5.075 μm, 56.082 ± 6.459 μm) is significantly larger than that of G. godefroyi (59.968 ± 3.484 μm, 45.439 ± 2.870 μm). Both 2C DNA content and pollen size are the effective characteristics for species discrimination. The reproductive evidence of high meiotic stability and normal pollen production indicate that both Gagnepainia species have high fertility and seed productivity, which are in accordance with the broad distribution. The present study provides good cytogenetic and pollen characters not only for plant identification, but also plant fertility assessment through plant genetic resource management and improvement of Gagnepainia.

Genome size analysis of field grown and somatic embryo regenerated plants in Allium sativum L.

In the present study, an efficient in vitro propagation protocol has been developed from clove explants of Allium sativum L., one of the oldest vegetable and medicinal plant used worldwide. Garlic is propagated vegetatively as cross-fertilization is strictly precluded due to sterile flowers. Due to a low rate of multiplication, limited genetic improvement possibility and increased germplasm degradation, plant tissue culture becomes an efficient and preferred tool for quality and rapid propagation of garlic. Here, the clove explants were cultured on Murashige and Skoog basal medium amended with different concentrations of Plant Growth Regulators (PGRs) namely 2,4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino purine (BAP), and 1-naphthalene acetic acid (NAA). Within 2weeks of inoculation, white compact callus was formed, maximum callus induction frequency (85.99%) was on 1.5mgl-1 2, 4-D added MS medium. Induced callus transformed into an embryogenic callus on 2, 4-D and BAP amended MS medium with highest embryogenic frequency (77.7%) was noted on 0.25mgl-1 2, 4-D and 1.0mgl-1 BAP added medium. Embryogenic callus differentiated into progressive stages of somatic embryos starting from globular, scutellar, and finally to coleoptilar stage of the embryo. Histological and scanning electron microscopic study of embryogenic callus was conducted, showing different stages of embryos, their origin and development, re-confirming somatic embryogenesis incidence in A. sativum. Green and mature somatic embryos were germinated and converted into plantlets on 0.5mgl-1 BAP amended MS medium. The in vitro regenerated plants were cultured separately in IBA and NAA supplemented media for root induction. The MS medium amended with 1.0mgl-1 IBA proved to be the best PGR treatment in inducing roots. The rooted plants were acclimatized and transferred ex vitro with about 87% survival rate. Cytological and flow cytometric analyses were performed to assess the genetic stability of in vitro regenerated plants. Cytological studies of in vitro regenerated plants showed 2n = 16 chromosome number and did not reveal any numerical variation in chromosomes. Flow cytometry was employed to measure the 2C DNA content of somatic embryo regenerated A. sativum plants and compared with in vivo grown garlic. The histogram peaks of relative 2C DNA content of in vitro regenerated plantlets were similar to the corresponding 2C DNA peak of in vivo grown plants. Flow cytometric 2C DNA content of embryo regenerated and field-grown A. sativum plants were the same, i.e., 33.45pg and 33.56pg, respectively, confirming genetic similarity. In conclusion, the present cytological and flow cytometric study suggest that the in vitro culture conditions are quite safe, did not encourage genetic alterations, and regenerants were "true to type."

Endocrine-Independent Cytotoxicity of Bisphenol A Is Mediated by Increased Levels of Reactive Oxygen Species and Affects Cell Cycle Progression.

Bisphenol A (BPA) is used for the production of plastics and epoxy resins, which are part of packaging materials for food and beverages, and can migrate into food and the environment, thus exposing human beings to its effects. Exposure to BPA has been associated with oxidative stress, cell cycle changes, and genotoxicity, and is mediated by its known endocrine-disrupting activity. Possible BPA cytotoxicity without mediation by estrogen receptors has been reported in the literature. Here, we show the toxic effects of BPA by live-cell imaging on the fission yeast Schizosaccharomyces pombe, an experimental model lacking estrogen receptors, which were in line with data from flow cytometry on intracellular oxidation (76.4 ± 14.4 and 19.4 ± 16.1% of fluorescent cells for BPA treatment and control, respectively; p < 0.05) as well as delay in cell cycle progression (after 90 min of experiment, 48.4 ± 4.30 and 64.6 ± 5.46% of cells with a 4C DNA content for BPA treatment and control, respectively; p < 0.05) upon exposure to BPA. These results strongly support the possibilities that BPA-induced cell cycle changes can be independent of estrogen receptors and that live-cell imaging is a powerful tool for genotoxic analysis.

Genome size and SCoT markers as tools for identification and genetic diversity assessment in Echinacea genus

The Echinacea (coneflower) genus includes herbaceous flowering species mainly used for medicinal purposes, as well as the ornamental plants. Morphological similarities between Echinacea species often lead to their incorrect identification and characterization. Genome size and SCoT markers were used for identification and establishing the taxonomic relationships within Echinacea genus. The analysis of the nuclear DNA content indicated, that the majority of Echinacea species possessed intermediate genomes, and only E. pallida has a large genome size. 2C DNA content ranged from 14.31 pg/2C (E. simulata) to 28.59 pg/2C (E. pallida). Based on flow cytometric measurements (FCM) of the nuclear DNA content it was possible to distinguish three coneflower species, E. simulata, E. pallida and E. sanguinea, confirming validity of the method for initial species identification. Whereas, SCoT-PCR analysis enabled the identification of all studied accessions. Nine primers revealed polymorphism both between species, as well as varieties of E. angustifolia and E. paradoxa, demonstrating the usefulness of SCoT primers for genetic diversity studies in this genus. The phylogenetic analysis confirmed, that Echinacea species are closely related and within the investigated accessions two main phylogenetic groups exist, with one species (E. purpurea) being not clustered to any of the groups. Two methods, FCM and SCoT-PCR, can be successfully applied in identification and taxonomic relationship assessment, as well as in determination of the genetic diversity of Echinacea genus. Moreover, this is the first report on genome size that includes eight Echinacea accessions.

Genome size evolution and chromosome numbers of species of the cryptanthoid complex (Bromelioideae, Bromeliaceae) in a phylogenetic framework

Abstract We describe the chromosome numbers and genome sizes of species of the cryptanthoid complex of Bromeliaceae in a phylogenetic framework and their relationship with habitat preferences. The 2C DNA contents varied 2.13-fold among species, ranging from 0.76 to 1.66 pg. A significant difference in DNA content was found among Cryptanthus, Hoplocryptanthus and Rokautskyia. Moreover, species from campos rupestres and the Atlantic Forest had lower and higher genome size values, respectively. The smaller genome sizes of Hoplocryptanthus spp. from campos rupestres may be related with the large genome constraint. The species show a highly conserved ploidy (with 2n = 32 and 34), although the genome sizes varied considerably. The observed variation in chromosome numbers seems to be influenced by dysploidy, but additional investigations are needed. Our study demonstrates that the genome size variation in the cryptanthoid complex species is not strictly related to the phylogenetic relationships and has probably been influenced by different evolutionary processes.

Rapid plant regeneration in industrially important Curcuma zedoaria revealing genetic and biochemical fidelity of the regenerants.

The present investigation was carried out to establish an efficient and reproducible micropropagation protocol for the production of morphologically, genetically and chemically uniform plants of Curcuma zedoaria. Axillary bud explants of C. zedoaria were inoculated into MS basal medium supplemented with various combinations and concentrations of 6-benzyladenine (2.2-22.2µM, BA), kinetin (2.3-23.2µM, Kin), indole-3-acetic acid (2.9-11.4µM, IAA), α-naphthalene acetic acid (2.7-10.2µM, NAA) and adenine sulphate (33.9-203.6µM, Ads). Almost 95% of rhizome buds sprouted on MS medium supplemented with 13.3μM BA, 5.7μM IAA and 63.9μM Ads giving rise to an average of 12.89 ± 0.02 shoots within 6weeks. However, the maximum number of roots (25.8 ± 0.07 roots per explant) was obtained on half strength MS medium supplemented with 7.4µM of IBA after 4weeks of inoculation. Morphological characteristics were similar in both conventionally propagated and micropropagated plants. Additionally, genetic homogeneity of in vitro plants was further confirmed through ISSR and flow cytometry analysis. A total of 27 ISSR primers were screened, out of which 13 ISSR primers generated 58 monomorphic and reproducible bands thereby confirming the genetic uniformity of obtained plants. The mean 2C DNA content of the mother plant (2.96pg) was similar to that of in vitro derived plants (3.07pg). Gas chromatography-mass spectrometry (GC-MS) analysis showed similarity in the qualitative profile of chemical constituents of essential oil and high-performance liquid chromatography analysis revealed no significant differences in curcumin content in the tissue culture regenerants and mother plants of C. zedoaria. Therefore, the present micropropagation protocol could be effectively employed to generate true to type plantlets of C. zedoaria.