In vitro induction of secondary xylem-like tracheary elements in calli of hybrid poplar (Populus sieboldii × P. grandidentata)

The formation of tracheary elements was induced in calli derived from petioles of hybrid poplar (Populus sieboldii × P. grandidentata) after 10 days of culture on medium that lacked auxin but contained 1 μM brassinolide. Some differentiated cells formed broad regions of cell walls and bordered pits, which are typical features of tracheary elements of secondary xylem. Other differentiated cells resembled tracheary elements of primary xylem, with spiral or reticulate thickening of cell walls. The tracheary elements that developed in calli were formed within cell clusters. This induction system provides a new model for studies of the mechanism of differentiation of secondary xylem cells in vitro.     

In vitro induction of tetraploids in cassava variety ‘Xinxuan 048’ using colchicine

Polyploids have great breeding value as they could have higher vegetative yields, higher qualities and greater tolerances against biotic and abiotic stresses. This research is focusing on in vitro colchicine-induced tetraploids in cassava. The survival rate of explants decreased with the increase of colchicine concentrations. Based on the survival rate, the treatment with 0.05 g l^?1 colchicine for 2 days was the best protocol for tetraploid induction in the cassava variety ‘Xinxuan 048’. The determination of ploidy levels showed that 28 autotetraploidy and 12 mixoploidy plantlets were obtained from 44 plantlets. Thus, 90.9% of the plants were variants. Significant differences in morphology and anatomy were found between the diploid and tetraploid plants. Tetraploid plants showed better photosynthetic capacities than the original diploid plants. The tetraploid cassava regenerated herein will enrich the germplasm spectrum and open a new arena for breeding novel triploids with elite cultivars by interploid crossing in the future.     

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.     

QTL analyses of fiber components and crude protein in an annual × perennial ryegrass interspecific hybrid population

Annual (Lolium multiflorum Lam.) and perennial (Lolium perenne L.) ryegrasses are two important forage and turfgrass species. Improving the digestibility of forage by decreasing fiber content is a major goal in forage crop breeding programs. An annual × perennial ryegrass interspecific hybrid population was used to map quantitative trait loci (QTLs) for fiber components, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL), and crude protein (CP). Samples were harvested three times in August and September 2003 and August 2004, respectively. Simple interval mapping was used to detect QTLs from both the male and female parental maps previously developed for the population. Fiber components were all correlated positively with each other and were negatively correlated with CP. The largest correlations were between NDF and ADF with r = 0.86, 0.72, and 0.82 for each of the three harvests. All four traits showed intermediate broad-sense heritability values ranging from 0.35 to 0.72. A total of 63 QTLs were detected for the four traits measured over the three harvests from both the female and male maps. Coincident QTLs were detected on linkage groups (LGs) 2, 6, and 7 for NDF, LGs 1, 2, and 7 for ADF, LGs 6 and 7 for ADL, and LG 2 for CP, respectively. Coincident QTLs were also detected on LGs 2, 6, and 7 for NDF and ADF, providing evidence of the genetic basis of the observed high level of phenotypic correlation. The QTLs on LGs 2, 6, and possibly 7 for fiber components were co-located on the same LG as several lignin biosynthetic genes from perennial ryegrass.     

Identification of quantitative trait loci controlling winter hardiness in an annual × perennial ryegrass interspecific hybrid population

Winter hardiness is a quantitative trait and the lack of it limits geographic distribution of ryegrass. Improving winter hardiness is an important breeding goal in ryegrass breeding programs. An understanding of the genetic basis for the component traits of winter hardiness would allow more efficient selection. A three-generation interspecific population of an annual × perennial ryegrass consisting of 152 progenies was used to map quantitative trait loci (QTL) that control winter hardiness-related traits including fall growth (FG), freezing tolerance (FT), and winter survival (WS) over 2 years. A total of 39 QTL were identified for the three traits from both the female parental (MFA) and the male parental (MFB) maps, of which 13 were for FG, 6 for FT, and 20 for WS. The proportion of phenotypic variation explained by individual QTL ranged from 10.4 to 22.1%. Both FG and FT were positively correlated with WS. Common QTL were detected between FG, FT, and WS. The QTL associated with WS on linkage groups (LGs) 4 and 5, and the QTL for FT on LG 5 were consistently identified over years and maps. These consistent QTL might serve as potential tools for marker-assisted selection to improve ryegrass winter hardiness.     

In vitro induction and characterization of hexaploid Pennisetum × advena,an ornamental grass

Plant Cell, Tissue and Organ Culture (PCTOC) - Pennisetum × advena (purple fountain grass) is an ornamental grass with considerable commercial value. In vitro induction of...     

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.     

Warm temperature accelerates short photoperiod-induced growth cessation and dormancy induction in hybrid poplar (Populus × spp.)

There is increasing evidence that temperature, in addition to photoperiod, may be an important factor regulating bud dormancy. The impact of temperature during growth cessation, dormancy development, and subsequent cold acclimation was examined in four hybrid poplar clones with contrasting acclimation patterns: ‘Okanese’—EARLY, ‘Walker’—INT1, ‘Katepwa’—INT2, and ‘Prairie Sky’—LATE. Four day–night temperature treatments (13.5/8.5, 18.5/13.5, 23.5/8.5, and 18.5/3.5°C) were applied during a 60-day induction period to reflect current and predicted future annual variation in autumn temperature for Saskatoon, SK. Warm night temperature (18.5/13.5°C) strongly accelerated growth cessation, dormancy development, and cold acclimation in all four clones. Day temperature had the opposite effect of night temperature. Day and night temperatures appeared to act antagonistically against each other during growth cessation and subsequent dormancy development and cold acclimation. Growth cessation, dormancy development, and cold acclimation in EARLY and LATE were less affected by induction temperature than INT1 and INT2 suggesting that genotypic variations exist in response to temperature. Separating specific phenological stages and the impact by temperature on each clone revealed the complexity of fall phenological changes and their interaction with temperature. Most importantly, future changes in temperature may affect time to growth cessation, subsequently altering the depth of dormancy and cold hardiness in hybrid poplar.     

Anatomy and morphology of photinia (Photinia × fraseri Dress) in vitro plants inoculated with rhizobacteria

Photinia × fraseri Dress (photinia) is a woody plant with high ornamental value. The anatomy and morphology of micropropagated photinia inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense and Azotobacter chroococcum, in combination with pulses of 49.2 μM indole-3-butyric acid during rhizogenesis, were characterized using light and electron microscopy. Leaves of inoculated in vitro plants showed better development than those subjected to auxin control only. All inoculated treatments, independent of the bacterial strain used, had leaves with two layers of palisade parenchyma, a thick cuticle and linear unicellular trichomes. There was no proliferation of undifferentiated tissue in any treatment and the plants showed shoot–root vascular connections. Ex vitro leaves and in vitro plants inoculated with Azospirillum brasilense Cd and Azotobacter chroococcum 42 had large stomata with elliptic aperture radially surrounded by small stomata on the abaxial foliar surface. In addition, plants of these treatments had a large root hair zone over the root surface. Bacteria were only observed on surfaces of root hairs. The results suggest that the structural changes induced by bacterial inoculation of photinia in vitro plants could lead to better adaptation to ex vitro conditions after transplanting.     

In vitro response of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora)

The aim of present investigation was to study the effect of storage conditions on percentage germination of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora). Different batches of encapsulated and non-encapsulated embryos were preserved at room temperature, 4°C, in liquid nitrogen as such and by embedding in liquid paraffin. In the encapsulated somatic embryos stored at room temperature in sealed Petri plates, percentage of germination was 24.99%, but 5.55% in non-encapsulated embryos after 3 days of storage. Encapsulated embryos stored in vials containing liquid paraffin at room temperature were germinated at 18.05% after 60 days of storage, while it was 13.88% in non-encapsulated embryos after 45 days of storage. Encapsulated somatic embryos stored at 4°C in sealed Petri plates remained viable for up to 75 days with 6.94% germination, whereas non-encapsulated embryos remained viable for up to 45 days with 24.99% germination. Encapsulated embryos stored at 4°C in vials filled with paraffin germinated at 11.11% after 120 days of storage, but 5.55% in non-encapsulated embryos after 90 days of storage. Encapsulated and non-encapsulated embryos stored in liquid nitrogen showed 58.33 and 51.38% survival, respectively, after 7 months of storage. The plantlets developed from these embryos were transplanted after acclimatization and are growing normal.     

In vitro anther culture of sweet orange (Citrus sinensis L. Osbeck) genotypes and of a C. clementina × C. sinensis ‘Hamlin’ hybrid

Citrus, and particularly sweet oranges, are very recalcitrant to anther culture. In this paper it was evaluated for the first time the response of 27 genotypes of Citrus sinensis and of one hybrid C. clementina × C. sinensis, to in vitro anther culture. Ten genotypes of sweet oranges showed embryogenic callus induction, mostly blood sweet oranges genotypes, such as Tarocco, Moro and Sanguinelli. In vitro microspore developmental switches from the gamethophytic to the sporophytic pathway were shown by DAPI staining in microspores of these responsive genotypes, after 10 months in culture. However, microsatellite marker analyses showed that these calli were heterozygous. The flow-cytometric analysis of these embryogenic calli showed the presence of two peaks, corresponding to haploid (n) and diploid (2n) genotypes. Differently, anther cultures of the hybrid C. clementina × C. sinensis produced tri-haploid (3n) embryogenic calli and the embryos obtained were homozygous when analyzed by molecular markers (sample sequence repeats), confirming the more responsive characteristic of clementine to microspore embryogenesis through anther culture.     

In vivo and in vitro function of human UDP-galactose 4'-epimerase variants

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4′-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4′-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4′-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.     

Microenvironmental influences of apoptosis in vivo and in vitro

The apoptosis program of physiological cell death elicits a range of non-phlogistic homeostatic mechanisms—“recognition, response and removal”—that regulate the microenvironments of normal and diseased tissues via multiple modalities operating over short and long distances. The molecular mechanisms mediate intercellular signaling through direct contact with neighboring cells, release of soluble factors and production of membrane-delimited fragments (apoptotic bodies, blebs and microparticles) that allow for interaction with host cells over long distances. These processes effect the selective recruitment of mononuclear phagocytes and the specific activation of both phagocytic and non-phagocytic cells. While much evidence is available concerning the mechanisms underlying the recognition and responses of phagocytes that culminate in the engulfment and removal of apoptotic cell bodies, relatively little is yet known about the non-phagocytic cellular responses to the apoptosis program. These responses regulate inflammatory and immune cell activation as well as cell fate decisions of proliferation, differentiation and death. Here, we review current knowledge of these processes, considering especially how apoptotic cells condition the microenvironments of normal and malignant tissues. We also discuss how apoptotic cells that persist in the absence of phagocytic clearance exert inhibitory effects over their viable neighbors, paying particular attention to the specific case of cell cultures and highlighting how new cell-corpse-clearance devices—Dead-Cert^® Nanoparticles—can significantly improve the efficacy of cell cultures through effective removal of non-viable cells in the absence of phagocytes in vitro.     

In vitro induction of tetraploid and resulting trait variation in Populus alba × Populus glandulosa clone 84 K

Plant Cell, Tissue and Organ Culture (PCTOC) - As a model plant, poplar 84 K (Populus alba?×?P. glandulosa) plays a key role in fundamental research in forest molecular...     

Evaluation of inhibitory effect and apoptosis induction of Zyzyphus Jujube on tumor cell lines,an in vitro preliminary study

In the present study, water extract of dried fruit of Zyzyphus Jujube was tested for its possible anticancer effect and induction of apoptosis on human tumor cell lines, HEp-2, HeLa and Jurkat cell lines. The inhibitory effect of water extract of this fruit on cell proliferation was assessed by MTT colorimetric assay. The induction of apoptosis of this extract was analyzed by DNA fragmentation analysis. Zyzyphus Jujube extract showed inhibitory effects on mentioned cell lines. Jurkat leukemic line was found the most sensitive cells with IC50 of 0.1 μg mL⁻¹. Our study also showed a typical DNA laddering in this cell line. The present study showed cytotoxic activity of Zyzyphus Jujube on tumor cells. Although Zyzyphus Jujube has useful compounds for medical applications.     

In vitro and in vivo antioxidant activity of exopolysaccharide fractions from Bifidobacterium animalis RH

The aim of the study was to purify the exopolysaccharides (EPS) produced from Bifidobacterium animalis RH, which was isolated from the feces of Bama centenarians in Guangxi of China, and evaluate their antioxidant activities in vitro and in vivo. 2 fractions, a neutral EPS fraction (EPSa) and an acidic EPS fraction (EPSb), were obtained and compared for antioxidative activity. In vitro, they both showed remarkable inhibition effect on lipid peroxidation and strong DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, in which the last two were measured by the electron spin resonance (ESR). In vivo, EPSa and EPSb were orally administrated for 30 days in a d-galactose induced aged mice model. As results, they both could significantly increase the activities of SOD, CAT and total antioxidant capacity (TAOC) in serums and glutathione GST in livers. They also could inhibit significantly the formation of MDA in serums and livers, and reduce the activity of MAO and lipofuscin accumulation in mice brain. Moreover, EPSb exhibited much higher antioxidant activities than EPSa in vitro and in vivo. The results suggested that EPS fractions of Bifidobacterium animalis RH had direct and potent antioxidant activities.     

In vitro release behavior and cytotoxicity of doxorubicin-loaded gold nanoparticles in cancerous cells

Doxorubicin (DOX), a common cancer chemotherapeutics, was conjugated to folate-modified thiolated-polyethylene glycol-functionalized gold nanoparticles. The in vitro, controlled release behavior of DOX-loaded gold nanoparticles was observed using porous dialysis membranes (cut-off = 2 kDa). DOX-loaded gold nanoparticles had higher cytotoxicity for folate-receptor-positive cells (KB cells) compared to folate-receptor-negative cells (A549 cells) which were 48 and 62% viable for 10 μM doxorubicin, respectively. This indicates the potential of these nano-carriers for targeted-delivery. In addition, healthy cell viability was 69% for 10 μM free doxorubicin whereas for the same content of drug in DOX-loaded nanoparticles healthy cell viability increased to 80%.