Assessment of polyembryony in lemon: rescue and in vitro culture of immature embryos

The 27 lemon cultivars analysed could be considered slightly or moderately polyembryonic, with 25 to 43% of seeds being polyembryonic and from 1.3 to 1.6 embryos per seed. On this basis, it is necessary to rescue zygotic embryos at an immature stage. Rescue and in vitro embryo development have been studied in two Citrus limon polyembryonic cultivars. Sucrose (50 and 70 g/l) was combined with Murashige and Skoog and Gamborg’s B5 media and tested for optimal growth response. An important effect of genotype was observed: embryos from cultivar ‘Eureka’ had greater survival, germination percentage, and radical development. While the sucrose concentration in the medium did not have an effect on germination, the medium affected the embryo survival and root development of the seedlings, Gamborg’s B5 medium giving the best results. The ability to form plants in vitro was affected by an increase of embryo developmental stage. The germination and seedling height were greater with embryos of seeds collected 135–150 days after anthesis.     

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm² undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.     

The isolation of tissue culture ofPopulus alba L. ‘Pyramidalis’

Tissue culture was isolated from the stem ofPopulus alba L. ‘pyramidalis’. Callus formation was observed since November till March (1974),i.e. till the formation of calluses suitable for further subeultivation. The most vigorous growth was obtained with the callus culture cultivated on the nutrient medium of DIAZ-COLONet al. (1972) on which more than 11 g of fresh matter was produced after 30 d at the end of the first year of cultivation in darkness, with inoculum weight 1.5-1.8 g. A mild decrease in growth rate of the tissue culture was observed after the first year of cultivation. When illuminated, the originally yellow calluses turned green. The morphological and anatomical structure of the callus culture is also described and cell shape and cell size evaluated.     

Genetic and biochemical studies on perchloroethylene ‘in vitro’ and ‘in vivo’

Perchloroethylene (PCE) was tested in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system (S9) and ‘in vivo’ by the intrasanguineous host-mediated assay. In addition, enzyme alteration studies were performed in mice non-pretreated or pretreated with phenobarbital + β-naphthoflavone. PCE did not induce any genetic effect either ‘in vitro’ or ‘in vivo’. In the suspension test, PCE was more toxic without metabolic activation and less toxic with mammalian microsomal activation. The enzymatic determinations showed an increase of the aminopyrine demethylase activity and of the level of cytochrome P-450.     

‘In vitro’ and ‘in situ’ decomposition of nuisance macroalgae Cladophora glomerata and Pilayella littoralis

The decomposition of two macroalgal species Cladophora glomerata (CHLOROPHYTA) and Pilayella littoralis (PHAEOPHYTA) was studied in the laboratory and field conditions. These species are known to cause the extensive macroalgal blooms in the whole coastal range of the Baltic Sea. The objective of the experiments was to determine decomposition rates of the macroalgae, follow the changes in tissue nutrient content and validate the role of benthic invertebrates in this process. In the laboratory conditions, the differences in the decomposition rates of the algae were mainly due to the oxygen conditions. The weight loss of C. glomerata was slightly higher in anaerobic conditions than in aerobic conditions. If 99% of initial dry weight of P. littoralis was lost in aerobic conditions then only 20% was lost in anaerobic conditions. In general, the loss of phosphorus and nitrogen in algal tissues followed the weight loss. As an exception, the amount of nitrogen changed very little during the decomposition of C. glomerata. In field conditions, the photosynthetic activity exceeded the decomposition rate of C. glomerata at lower temperatures in spring. The decomposition of P. littoralis was estimated at 49% of its initial dry weight. The addition of benthic invertebrates had no effect on the decomposition process. In summer, the decomposition rates were estimated at 65% for C. glomerata and 68% for P. littoralis being in the same order of magnitude as observed in laboratory conditions. If the decomposition of C. glomerata was faster at the end of the experiment, the most significant losses of weight of P. littoralis took place during the first 2 weeks of deployment. Idotea baltica significantly contributed to the loss of C. glomerata. The decomposition rate of P. littoralis was reduced by the presence of Mytilus edulis and increased by Gammarus oceanicus.     

Demonstration of ‘cardiac-specific’ myosin heavy chain in masticatory muscles of human and rabbit

Summary Human and rabbit masticatory muscles were analyzed immuno-and enzyme-histochemically using antibodies specific to cardiac , slow and fast myosin heavy chain isoforms. In human masseter, temporalis, and lateral pterygoid muscle cardiac myosin heavy chain is found in fibres that contain either fast, or fast and slow myosin heavy chain. In rabbit masseter, temporalis and digastric muscles, fibres are present that express cardiac myosin heavy chain either exclusively, or concomitantly with slow myosin heavy chain or fast myosin heavy chain. Our results demonstrate a much broader distribution of cardiac myosin heavy chain than hitherto recognized and these might explain in part the specific characteristics of masticatory muscles. The cardiac myosin heavy chain is only found in skeletal muscles originating from the cranial part of the embryo (including the heart muscle) suggesting that its expression might be determined by the developmental history of these muscles.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Leaf anatomy and water loss of in vitro PEG-treated ‘Valiant’ grape

Polyethylene glycol was used to induce water stress of micropropagated Valiant grape. Reduced growth and slow rooting were observed in treated plantlets with 2, 4 and 6% polyethylene glycol as compared to control plantlets with no polyethylene glycol in the rooting medium. At high concentrations of 4 and 6%, leaves exhibited wilting and necrosis. At the 2% level, plantlets recovered and grew satisfactorily. Detached leaves of treated plantlets with 2% polyethylene glycol lost less water than controls when exposed to low humidity for 4 hours. Leaf anatomy of plantlets treated with 2% polyethylene glycol, control (in vitro plantlets) and greenhouse-grown plants were compared under light microscopy. Leaves from control plantlets contained larger mesophyll cells, lacked normal palisade layer formation, had greater intercellular pore spaces and fewer chloroplasts. Leaves from polyethylene glycol-treated plantlets, however, had smaller mesophyll cells, a more defined palisade layer, reduced intercellular pore space and the greatest number of chloroplasts. These results suggest that an osmoticum such as polyethylene glycol may be used to induce more normal leaf anatomy and reduced water loss in micropropagated Valiant grapes.Abbreviations BA 6-benzylaminopurine - FAA formalin-acetol-alcohol - MS Murashige & Skoog (1962) medium - MW molecular weight - NAA napthaleneacetic acid - PEG polyethylene glycol - TBA tertiary butyl alcohol     

Improvement of in vitro propagation of apricot cultivar ‘Bebecou’

Factors affecting successful establishment in vitro, rapid proliferation and rooting of apricot cultivar ‘Bebecou’ were studied. Ethanol and NaOCl were applied in several combinations for disinfection; chilling, plant growth regulators BA, IAA and GA₃, antibiotics, different culture vessels and systems of subculture were evaluated for the optimization of shoot proliferation and the auxins NAA and IBA were assessed for root induction. The highest number of new microshoots/explant (18.7) was obtained in a culture medium supplemented with 2.2 μM BA+0.57 μM IAA after 300 h of chilling. The effect of GA₃ (11.4 μM) on shoot proliferation was positive in combination with 4.4 or 8.9 μM BA. Shoot length and productivity were highest at 2.2 μM BA+11.4 μM GA₃+0.57 μM IAA and at 2.2 μM BA+0.57 μM IAA, respectively and decreased as cytokinin concentration increased. The antibiotic ‘Na-cefotaxime’ had a minimal impact on shoot growth when used at the lowest concentration (250 mg l⁻¹). Subculture every 2 weeks in a medium supplemented with 2.2 μM BA and 0.57 μM IAA was more efficient for shoot induction than alternation of 20 days culture in a propagation medium supplemented with 2.2 μM BA and 10 days culture in an elongation medium supplemented with 1.1 μM BA and 5.71 μM IAA. The highest number of roots/shoot (8.1) was recorded at 19.6 μM IBA.     

In vitro haploid and dihaploid production via unfertilized ovule culture

Haploids and doubled haploids are very important in plant breeding, enabling the time needed to produce homozygous lines to be shortened compared with conventional breeding. In the present review, emphasis is given to haploid induction through unfertilized ovule/ovary culture. Attention is given to induction of haploid plants from female gametophyte culture through analysis of factors in the processes of gynogenesis, including genotype selection, stage of ovule development, pretreatment, and culture media containing nutritional components and phytohormones. The gynogenetic approach may be of great value in discovering novel genetic recombinations. Application of double haploids in genetics and plant breeding is also highlighted. This review also identifies some existing knowledge gaps where work may increase the efficiency of this process in different plant species.     

Cell division study in ‘pollen mother cells’ and ‘pollen grains’ of in vivo and ex vitro plants in Drimiopsis botryoides

The various normal and abnormal stages of meiosis and pollen mitosis of Drimiopsis botryoides are described, and a comparison between naturally propagated in vivo and tissue culture derived ex vitro plants in respect to their cytological behaviour presented. We also describe the floral morphology and investigate the relationship between the floral developmental stages and the progression of microgametogenesis. In total, 33 bivalents are observed in diakinesis, which indicate the diploid number 2n = 66 and this number is cross-checked by a haploid set of n = 33 chromosomes in pollen mitosis. Only 6.8% and 4.9% meiotic abnormalities were recorded on in vivo and ex vitro plants, respectively, which led to the formation of non-viable pollen. Finally, the microspores have to develop into tri-cellular male gametophyte. Only 0.2% pollen grains are found with a micro-nucleus. Though the higher pollen viability was recorded on both in vivo (89.3 ± 4.1%) and ex vitro (92.1 ± 4.6%) plants, but surprisingly the pollen germination rate is extremely low with 13.6 ± 1.74% and 21.3 ± 1.55%, respectively. The present study obviously enriches the cytological database of D. botryoides and may help future research on androgenesis and genetic improvement.     

XCI in preimplantation mouse and human embryos: first there is remodelling…

Female eutherians silence one of their X chromosomes to accomplish an equal dose of X-linked gene expression compared with males. The mouse is the most widely used animal model in XCI research and has proven to be of great significance for understanding the complex mechanism of X-linked dosage compensation. Although the basic principles of XCI are similar in mouse and humans, differences exist in the timing of XCI initiation, the genetic elements involved in XCI regulation and the form of XCI in specific tissues. Therefore, the mouse has its limitations as a model to understand early human XCI and analysis of human tissues is required. In this review, we describe these differences with respect to initiation of XCI in human and mouse preimplantation embryos, the extra-embryonic tissues and the in vitro model of the epiblast: the embryonic stem cells.     

Reversible and irreversible damage in reoxygenated ‘ischemic’ ventricular myocytes in culture

The LDH release pattern from cardiomyocytes under ischemia-like conditions shows two phases. In the initial slow phase, reoxygenation immediately stops further enzyme release. Accelerated LDH release, which occurs concomitantly with Iysosomal enzyme release, characterizes the second phase of ischemia. Reoxygenation at this stage does not put a stop to further enzyme release.Reoxygenation during the first phase of ischemia rapidly restored ATP level, while in the second phase, ATP levels remained low even after 6 h of reoxygenation.This study as well as previous data seem to suggest that irreversible cellular damage leading to cell death, occurs by synergistic action of many effectors, each of which does not necessarily cause irreversible damage.     

Phenomenon of “Siamese embryos” in cereals in vivo and in vitro: Cleavage polyembryony and fasciations

The genesis of wheat microsporial polyembryoids in vitro was analyzed in detail. The nature of different phenotypes of cereal polymeric embryos was identified. They represent the class “multiple shoot meristems,” which results from a cleavage polyembryony and is accompanied by organ fasciations of all known types (radial, flat, or ring). The morphological nature of cereal embryonic organs has been clarified: shoot meristem—axial organ; scutellum—lateral outgrowth of this axis; coleoptile—derivative of shoot meristem but fused with scutellum; terminality of scutellum—the result of linear fasciation that occurred historically. An explanation is given on how the structural model of an auxin polar transport works during the establishment of bilateral symmetry in a cereal embryo that is associated with the inverted polarization of the carrier protein PIN1 on cell membranes and, correspondingly, with the inverted auxin transport performed by this carrier (Fischer-Iglesias et al., 2001; Forestan et al., 2010).     

In vitro culture of vanhoutte's spirea explants from ‘secondary cultures’ and dormant stems forced in solutions containing plant growth regulators

Explants from new growth of forced dormant stems and secondary cultures of Vanhoutte's spirea were cultured on Linsmaier and Skoog (L.S.) medium containing benzyladenine (BA), indoleacetic acid (IAA), thidiazuron (TDZ), and zeatin. The dormant stems were forced by immersing their basal portions in forcing solutions containing 626 µM 8-hydroxyquinoline citrate (8-HQC) and 2% sucrose. BA and gibberellic acid (GA₃) were also added into the forcing solutions to determine if explants obtained from the new growth will benefit from this treatment when culturedin vitro.L.S. medium supplemented with 5 µM BA alone, 5 µM BA plus 1 or 5 µM IAA, and 0.5 or 0.75 µM TDZ alone produced the best shoot proliferation for both sources of explants. BA and GA₃ appeared to be taken up from the forcing solution by the new softwood growth. BA in the forcing solution stimulatedin vitro shoot proliferation in different degrees depending on the period of treatment, while GA₃ caused lessin vitro shoot proliferation. It is proposed that forcing solutions containing plant growth regulators (P.G.R.) are a useful approach for manipulating responses of plant tissues culturedin vitro.     

Cloning and characterization of chicken α5 integrin: Endogenous and experimental expression in early chicken embryos

Key roles for fibronectin and its integrin receptors have been postulated in the multiple cell-matrix interactions essential for chick embryo morphogenesis. However, mechanistic studies of these processes have been hampered by the current absence of sequence data and chicken cDNA clones for the major fibronectin receptor subunit, integrin α5 (ITGA5). We report here the sequence, endogenous expression pattern, and transfection of full-length chicken integrin α5. During early chicken embryonic development, α5 is highly expressed in cranial neural folds and migrating neural crest cells, suggesting potential roles in neural crest formation and migration. In fact, over-expression of this integrin in early neural tube selectively induces BMP5, a growth factor recently implicated in neural crest formation. Availability of these α5 integrin tools should facilitate studies of its functions in early embryonic development.