Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Superovulation and embryo collection in nulliparous Boer goat does immunized against a recombinant ovine alpha-subunit inhibin

With the purpose of eliciting a superovulatory response, 12 adult nulliparous Boer goat does were actively immunized against a recombinant a-subunit of ovine inhibin (roIHN-alpha; two injections of 100 mg 4 weeks apart). Another 12 control Boer goat does were treated with physiological saline and acted as controls. One year later the immunized animals were boostered by the administration of another dose (100 mg) of the immunogen. Following treatment, blood samples were collected twice weekly for the periods of 16 and 12 weeks, respectively, to monitor the inhibin binding ability with the aid of a radio-tracer binding assay. Throughout the experiment, estrus detection was conducted twice daily with the aid of an aproned intact buck. From the first day after treatment to 48 h after standing estrus, ovarian activity was monitored daily by transrectal ultrasonography. On alternate estrous cycles, does were mated and 6 days later flushed transcervically to recover embryos. All goats treated with the roIHN-alpha produced antibodies reactive to the native bovine inhibin tracer-the titre increasing from 2.9 +/- 0.4 to a maximum of 21.9 +/- 2.9% binding after the second injection. The antibody titre gradually subsided over the next 16 weeks. The booster injection restored an elevated antibody titre (11.7 +/- 0.4%), which was maintained until the end of the sampling period 12 weeks later. In the control goats only trace amounts of antibody were recorded throughout the trial. In the roIHN-alpha-immunized goats the number of follicles reaching a diameter of > 4 mm was 14.6 +/- 1.2 per doe. A positive correlation was recorded between the follicle number and antibody titre (r=0.61; P < 0.01). The number of follicles ovulating per doe (6.9 +/- 0.7) followed the same tendency-however, the proportion decreased with increasing follicle numbers. A relatively weak correlation was recorded between the inhibin binding ability and number of ovulations (r=0.27; P < 0.05). In the control goats the majority (92%) of follicles exceeding 4 mm in diameter ovulated (2.5 +/- 0.1 follicles/doe). Embryo collection proved unsatisfactory (42% versus 39% recovery for immunized and control animals, respectively)-presumably because the uterine lumen of the nulliparous does was too narrow to permit effective flushing. In the group of immunized goats the occurrence of short estrous cycles (< 15 days) recorded was 34% versus only 6% in the controls. Overall, immunization of goats against roIHN-alpha led to an almost six-fold increase in number of ovarian follicles, a three-fold increase in ovulations and, despite the low recovery rate, a more than three-fold increase in ova or embryos recovered. It may be concluded that treatment of female goats with roIHN-alpha leads to an inhibin antibody response, accompanied by enhanced ovarian activity. The response was, however, accompanied by a large proportion of retained follicles and a high incidence of short estrous cycles. These problems need to be further investigated before rendering the method fit for application in embryo transfer programs in goats. (C) 2007 Elsevier B.V. All rights reserved.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Wheat seed embryo excision enables the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Microbial impacts on plant-herbivore interactions: the indirect effects of a birch pathogen on a birch aphid

The role of indirect interactions in structuring communities is becoming increasingly recognised. Plant fungi can bring about changes in plant chemistry which may affect insect herbivores that share the same plant, and hence the two may interact indirectly. This study investigated the indirect effects of a fungal pathogen (Marssonina betulae) of silver birch (Betula pendula) on an aphid (Euceraphis betulae), and the processes underpinning the interaction. There was a strong positive association between natural populations of the aphid and leaves bearing high fungal infection. In choice tests, significantly more aphids settled on leaves inoculated with the fungus than on asymptomatic leaves. Individual aphids reared on inoculated leaves were heavier, possessed longer hind tibiae and displayed enhanced embryo development compared with aphids reared on asymptomatic leaves; population growth rate was also positively correlated with fungal infection when groups of aphids were reared on inoculated branches. Changes in leaf chemistry were associated with fungal infection with inoculated leaves containing higher concentrations of free-amino acids. This may reflect a plant-initiated response to fungal attack in which free amino acids from the degradation of mesophyll cells are translocated out of infected leaves via the phloem. These changes in plant chemistry are similar to those occurring during leaf senescence, and are proposed as the mechanistic basis for the positive interaction between the fungus and aphid.

Developmental changes in the germinability, desiccation tolerance, hardseededness, and longevity of individual seeds of Trifolium ambiguum

Background and Aims: Using two parental clones of outcrossing Trifolium ambiguum as a potential model system, we examined how during seed development the maternal parent, number of seeds per pod, seed position within the pod, and pod position within the inflorescence influenced individual seed fresh weight, dry weight, water content, germinability, desiccation tolerance, hardseededness, and subsequent longevity of individual seeds. Methods: Near simultaneous, manual reciprocal crosses were carried out between clonal lines for two experiments. Infructescences were harvested at intervals during seed development. Each individual seed was weighed and then used to determine dry weight or one of the physiological behaviour traits. Key Results: Whilst population mass maturity was reached at 33–36 days after pollination (DAP), seed-to-seed variation in maximum seed dry weight, when it was achieved, and when maturation drying commenced, was considerable. Individual seeds acquired germinability between 14 and 44 DAP, desiccation tolerance between 30 and 40 DAP, and the capability to become hardseeded between 30 and 47 DAP. The time for viability to fall to 50 % (p50) at 60 % relative humidity and 45 °C increased between 36 and 56 DAP, when the seed coats of most individuals had become dark orange, but declined thereafter. Individual seed f. wt at harvest did not correlate with air-dry storage survival period. Analysing survival data for cohorts of seeds reduced the standard deviation of the normal distribution of seed deaths in time, but no sub-population showed complete uniformity of survival period. Conclusions: Variation in individual seed behaviours within a developing population is inherent and inevitable. In this outbreeder, there is significant variation in seed longevity which appears dependent on embryo genotype with little effect of maternal genotype or architectural factors.

Germin, a protein marker of early plant development, is an oxalate oxidase

Germin is a homopentameric glycoprotein, the synthesis of which coincides with the onset of growth in germinating wheat embryos. There have been detailed studies of germin structure, biosynthesis, homology with other proteins, and of its value as a marker of wheat development. Germin isoforms associated with the apoplast have been speculated to have a role in embryo hydration during maturation and germination. Antigenically related isoforms of germin are present during germination in all of the economically important cereals studied, and the amounts of germin-like proteins and coding elements have been found to undergo conspicuous change when salt-tolerant higher plants are subjected to salt stress. In this report, we describe how circumstantial evidence arising from unrelated studies of barley oxalate oxidase and its coding elements have led to definitive evidence that the germin isoform made during wheat germination is an oxalate oxidase. Establishment of links between oxalate degradation, cereal germination, and salt tolerance has significant implications for a broad range of studies related to development and adaptation in higher plants. Roles for germin in cell wall biochemistry and tissue remodeling are discussed, with special emphasis on the generation of hydrogen peroxide during germin-induced oxidation of oxalate.

Difference in competence for in vitro proliferation and ex vitro growth of genetically identical mature and juvenile clones of apomictic Malus species

The apomictic system in Malus wits Used Is a model to examine rejuvenation by generating genetically identical tissue culture lines that had two entirely different developmental origins: either embryo-derived tissues (juvenile clones) or somatic tissue from the adult/mature tree (mature clones). These two lines were then subsequently used to examine in vitro difference between mature (M) and juvenile (J) tissues in potential for shoot, root proliferation and ex vitro (glasshouse) growth. The M clones of M. hupehensis and M. toringoides in vitro had significantly fewer total shoots and shoot more than 2 cm in length per proliferating explant than the J clones and also rooted less efficiently. Ex vitro (glasshouse) juvenile clones had shorter internodes, a greater number of leaves and more dry weight compared to their mature counterparts.

Effects of desiccation on seed germination, cracking, fungal infection and survival in storage of Sterculia foetida L

Seeds of Sterculia foetida were tested for germination following desiccation and subsequent hermetic storage. Whereas seeds at 10.3% moisture content were intact and provided 98% germination, further desiccation reduced germination substantially. The majority of seed coats had cracked after desiccation to 5.1% moisture content. Ability to germinate was not reduced after 12 months' hermetic storage at 10.3% and 7.3% moisture content at 15 degrees C or -18 degrees C, but was reduced considerably at 5.1%. Fungal infection was detected consistently for cracked seeds in germination tests and they did not germinate. However, almost all embryos extracted from cracked seeds germinated if first disinfected with sodium hypochlorite (1%, 5 minutes). In addition. 80 -100% of disinfected extracted embryos from cracked seeds stored hermetically for 28 d at -18 degrees C or -82 degrees C with 3.3% to 6.0% moisture content, and excised embryos stored in this way, were able to germinate. Hence. failure of the very dry seeds of Sterculia foetida to germinate was not due to embryo death from desiccation but to cracking increasing susceptibility to fungal infection upon rehydration. Cracking was associated negatively and strongly with relative humidity and appears to be a mechanical consequence of substantial differences between the isotherms of whole seeds compared with cotyledons and axes.

Patents and Haploid Plants

One of the important themes in any discussion concerning the application of haploids in agricultural biotechnology or elsewhere is the role of Intellectual Property Rights (IPR). This term covers both the content of patents and the confidential expertise, usually related to methodology and referred to as "Trade Secrets". This review will explain the concepts behind patent protection, and will use the international patent databases to analyse the content of these patents and trends over the last 20 years. This analysis from regions including North America, Europe, and Asia reveals a total of more than 30 granted patents and a larger number of applications. The first of these patents dates from 1986, and although the peak of activity was in the late 1990s, there has been continuous interest to the present day. The subject matter of these patents and applications covers methods for anther and pollen culture, ovule culture, the use of specific haploid-inducing genes, the use of haploids as transformation targets, and the exploitation of genes that regulate embryo development. The species mentioned include cereals, vegetables, flowers, spices and trees.

Use of secondary somatic embryos promotes genetic fidelity in cryopreservation of cocoa (Theobroma cacao L.)

The inability to conserve cocoa (Theobroma cacao L.) germplasm via sced storage and the vulnerability of field collections make the establishment of cryopreserved genebanks for the crop a priority. An effective encapsulation-dehydration based cryopreservation system has been developed for cocoa but because the somatic embryos used for freezing arise after a protracted period of callus culture there is concern about maintenance of genetic fidelity during the process. Microsatellite markers for seven of the 10 cocoa linkage groups were used to screen a population of 189 primary somatic embryo-derived emblings and the 43 secondary somatic embryos they gave rise to. Of the primary somatic embryos, 38.1% exhibited polymorphic microsatellite profiles while for secondary somatic embryos the frequency was 23.3%. The same microsatellite markers used to screen another population of 44 secondary somatic embryos cryopreserved through encapsulation-dehydration revealed no polymorphisms. Scanning electron microscopy showed the secondary somatic embryos were derived from cotyledonary epidermal cells rather than callus. The influence of embryo ontogeny on somaclonal variation is discussed.

Influence of freezable/non-freezable water and sucrose on the viability of Theobroma cacao somatic embryos following desiccation and freezing

Encapsulated cocoa (Theobroma cacao L.) somatic embryos subjected to 0.08-1.25 M sucrose treatments were analyzed for embryo soluble sugar content, non-freezable water content, moisture level after desiccation and viability after desiccation and freezing. Results indicated that the higher the sucrose concentration in the treatment medium, the greater was the extent of sucrose accumulation in the embryos. Sucrose treatment greatly assisted embryo post-desiccation recovery since only 40% of the control embryos survived desiccation, whereas a survival rate of 60-95% was recorded for embryos exposed to 0.5-1.25 M sucrose. The non-freezable water content of the embryos was estimated at between 0.26 and 0.61 g H2O g(-1)dw depending on the sucrose treatment, and no obvious relationship could be found between the endogenous sucrose level and the amount of non-freezable water in the embryos. Cocoa somatic embryos could withstand the loss of a fraction of their non-freezable water without losing viability following desiccation. Nevertheless, the complete removal of potentially freezable water was not sufficient for most embryos to survive freezing.

The long-term effect of active immunization against inhibin in goats

This experiment addresses the long-term effect of active immunization of goats against a recombinant ovine inhibin alpha subunit (roIHN-alpha). In late anestrus 100 mu g of roINH-alpha was administered to 40 pluriparous Boer goat does, followed, 4 weeks later, by a booster injection. Weekly blood samples were drawn to monitor the inhibin binding capacity with the aid of a radio-tracer binding assay. From the onset until 48 h after the end of each estrus, follicular development and ovulation rate were monitored at 24 h intervals by transrectal ultrasonography. Beginning in August and continuing into January, does were mated at every other estrus, and submitted to transcervical embryo collection. Seven months after the first immunization, the does were mated again and permitted to carry to term. All immunized does produced inhibin antibodies, an elevated titre being first detected 2 weeks after primary immunization. Maximum titres were reached after 6 weeks, i.e. 2 weeks after the booster injection. Thereafter, in the course of the following 32 weeks, the titre subsided gradually. The does started cycling by mid-August. At that stage the average number of follicles more than 4 mm in diameter, ovulations and total embryos and ova recovered were 14.7 (+/- 2.3), 5.3 (+/- 0.7) and 4.4 (+/- 1.0), respectively. A steady decline followed and in January the corresponding means were: 5.2 (+/- 0.6) follicles, 3.1 (+/- 0.6) ovulations and 1.2 (+/- 0.4) embryos and ova recovered. When mated toward the end of the breeding season, 85% of the does became pregnant to the first mating and 73% went to term. Healthy kids were born, the average litter size being 2.2 (+/- 0.1). In conclusion, immunization of goats against a recombinant inhibin alpha-subunit proved to be a practicable means of producing embryos for transfer purposes. After about half a year, when the inhibin antibody titre has subsided, it is possible to return the does to the breeding flock without risking complications with normal breeding activity. (c) 2009 Elsevier Inc. All rights reserved.