The present study describes an analysis of genotype and allele distribution at the porcine GH locus among day-10 pig embryos. Embryos were collected post mortem from 6 crossbred (Danish Landrace ? Yorkshire) sows inseminated with mixed Duroc semen and individually frozen for later analysis. After extraction, DNA was subjected to PCR amplification and restriction analysis with Msp I and Hae II enzymes. The genotype frequencies were: Msp I CD 0.17, DD 0.83; and Hae II AA 0.33, AB 0.58; and BB 0.09. The Msp I CC genotype was not found among analysed embryos. To our knowledge, this is the first report on the genotype and allele distribution at the GH locus among early pig embryos.
In this paper, the authors made an attempt to summarize the present knowledge on apoptosis in mammalian oocytes and embryos. On the contrary to necrosis, apoptosis is a programmed death of damaged or mutated cells. Several studies showed that it takes place during oogenesis (oocyte growth and maturation), as well as at some stages of preimplantation embryo development (morula, blastocyst). Although apoptosis is observed in vivo, the frequency of this phenomenon increases in vitro. There is a number of methods detecting apoptotic cells, however, none of them gives a clear evaluation of the studied process. A complex analysis with the use of several methods is therefore advised. Because in ovary the majority of oocytes undergo atresia (over 99%), the scientists concentrate on developing new methods aiming at improvement of females' reproductive performance. The process of apoptosis seems to be a crucial limiting factor.
This paper presents current methods of embryo and oocyte cryoconservation in the following species: mice, rabbits, sheep and goats, pigs, horses and cows. Both the freezing and vitrification methods are discussed with special emphasis on the major factors affecting the efficiency of these two methods.
The effect of sucrose and maltose in culture media PII, C17 and MN6 on androgenetic embryo formation was investigated in seven F2 triticale hybrid progenies. In all genotypes, the highest number of androgenetic embryos (89.8-320.6/100 anthers) was formed on medium C17 containing maltose. Also the highest number of green plants (6.17/100 embryos) was regenerated from embryos obtained on PII with sucrose. The effect of the physical environment (temperature, light) in the first week of embryo culture on the regeneration medium was tested. The highest rate of green plants per 100 embryos (2.5-11.8) was obtained from incubation at 22oC in the dark. Key words: androgenetic embryos, incubation temperature, media, plant regeneration, triticale.
Suboptimal conditions of oocyte maturation and embryo culture in vitro reduce their developmental competence as compared to in vivo development. It is well known now that some IVF blastocysts, despite a proper morphology, are not able to hatch and implant after transfer. Therefore, it is suggested that during in vitro culture embryos show an ability to adjust to suboptimal conditions, which is however accompanied by the changes in gene expression pattern resulting in reduced quality. Although gene expression analysis, as an invasive method, is considered as an indirect way of embryo quality evaluation, it provides valuable data on the processes crucial for growth and development.
In vitro culture of hybrid embryos and ovules from crosses of B. campestris, B. oleracea, B. fruticulosa and B. napus were conducted on White, Murashige and Skoog, Murashige and Skoog modified by Keller and Nitsh and Nitsh media. The highest efficiency of crosses was obtained in the crosses B. campestris x B. oleracea, and the lowest in crosses with B. fruticulosa. In cultures of 572 embryos from 11 cross combinations, plants were regenerated from 24,5% of cultured embryos. From 3 cross combinations 502 ovules were placed on the medium. In this case the plants were regenerated from 2 of 3 crosses and the efficiency of the culture was over 46%.
In intergeneric and interspecific crossing frequently occurs inhibition in the development of as a result of . Hence the necessity of in vitro culture of immature embryos. The paper presents attempts to obtain wheat plants through the culture of ovules isolated 1-4 days after pollination and 5-9 day embryos. The medium B5 without 2,4-D wads applied. Plants were obtained from the ovules and 7-9 day embryos, whereas 5-6 day embryos gave no plants. All obtained plants developed directly from embryos; no callus formation was observed.
The term "karyomere"designates a p[articular kind oforganization of the telophase nucleus, in which each individual chromosome is surrounded by a typical nuclear envelope, thus representing a small nuclear structure.In T.bielenensis they are formed during firts prophase and subsequently change into chromosomes making a metaphase plate.Each metaphase chromosome is completly enclosed by a double envelope, similar to a nuclear one but devoid of nuclear pores.The only place on the chromosome where the envelope is not complete is the region of the kinetochore.Anaphase starts with the division of the chromosome into chromatids and subsequent decondensation of the latter.During anaphase, te process of decondensation of the chromatids starts at the leading ends of each chromatid and hence, they acquire a tennis racket-like appearance.As a result, on the spindle poles two groups of the chromosome vesicles or karyomeres are gethered.Each karyomere has the appearance of a typical nucleus enclosed by a double envelope pierced by nucleus pores filed with nucleoplasm containing patches ofchromatin.It is suggested that karyomeres are formed only in species with a large amount of chromatin in their nuclei.
Bone marrow (BM) was for many years primarily regarded as the source of hematopoietic stem cells. In this review we discuss current views of the BM stem cell compartment and present data showing that BM contains not only hematopoietic but also heterogeneous non-hematopoietic stem cells. It is likely that similar or overlapping populations of primitive non-hematopoietic stem cells in BM were detected by different investigators using different experimental strategies and hence were assigned different names (e.g., mesenchymal stem cells, multipotent adult progenitor cells, or marrow-isolated adult multilineage inducible cells). However, the search still continues for true pluripotent stem cells in adult BM, which would fulfill the required criteria (e.g. complementation of blastocyst development). Recently our group has identified in BM a population of very small embryonic-like stem cells (VSELs), which express several markers characteristic for pluripotent stem cells and are found during early embryogenesis in the epiblast of the cylinder-stage embryo.
Using the regeneration method described by Feldman and Marks (1986), the level of somaclonal variation (frequency of gene mutations and chromosomal changes) was determined in A. thaliana regenerants from root and shoot explants. The explants were regenerated via indirect organogenesis with callus stage. The level of embryo lethal mutations and changes in chromosome number were estimated in R2 generative progeny of regenerants, and confirmed in R3 generation. The frequency of R1 plants carrying gene mutations was similar for all types of explants and on average it reached 8.3%, however only 47% of R1 plants showed the presence of the same embryo mutation in R2 and R3 generation. The frequency of embryo-lethal mutants, among 25 571 embryos tested, was 1.7%, while 0.08% embryos carried chlorophyll mutations. The plants with increased chromosome number were observed in the progeny of 32.2 % R1 regenerants, i.e. with eight times higher frequency than regenerants carrying gene mutations. All polyploid plants were obtained from shoot explants, while regenerants from roots carried normal diploid number of chromosomes. The observed changes in chromosome number were discussed in relation to the different content of DNA in root and shoot cells of A. thaliana.
The influence of cultivar, donor plant and culture procedure on the efficiency of androgenesis was studied in carrot anther culture. Experiments were carried out on five carrot cultivars: CxC 9900 F1, Lucky B F1, HCM, Beta III and Perfekcja, which were chosen because of their high carotene contents. Two procedures of anther culture were compared: (1) incubation in darkness for two weeks, followed by exposure to continuous light and transfer onto a fresh medium of the same composition; and (2) incubation in darkness until embryos appeared, without transfer onto a fresh medium. Temperature was +27oC all the time. Genotype played an important role in the process of androgenesis in carrot anther culture.The efficiency was the highest in cv. HCM ? 5.6 embryos per 100 anthers. Considerable differences in the capacity for androgenesis were observed between individual donor plants. The ratio of embryos obtained per 100 anthers for cv. HCM varied from 0.0 to 48.9. The second procedure of anther culture proved to be more efficient, cheaper and less complicated.
The interspecific F1 (A. galanthum x A. cepa) hybrids were obtained via embryo rescue technique. For hybrids and parental plants, the morphological traits and pollen fertility evaluation were made. The tested F1 hybrids were similar to the maternal form regarding to the leaves' length, while the number of bulbils leaves and stem diameter were similar to the paternal form. Pollen viability test showed that F1 hybrid plants exhibited complete or partial male sterility. Cytological observations of microsporogenesis and tetrads revealed no disturbances in meiosis; however, the presence of dyads, tryads (22%) and tetrads degeneration (1%) was recorded. The analyses of chloroplast DNA proved that all F1 progenies examined possessed the cytoplasm of A. galanthum. We concluded that the sterility or low pollen viability of F1 hybrids is caused by the presence of galanthum cytoplasm.
The purpose of this paper is to review information regardind the co-ordination of nuclear and cytoplasmic events during embryo reconstruction, in particular the direct and indirect effects of maturation (meiosis) mitosis promoting factor (MPF), upon the transferred nucleus.These will be discussed in relation to DNA replication, the maintenance of correct ploidy, the occurence is primarilu concerned with the reconstruction of mammalian embryos, specific examples from amphibians will also be cited.
The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta -galactosidase (beta -gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were coinjected with the scaffold attachment sequences - SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta -galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta -galactosidase expression. The percentage of transgenesis with pCMV-lacZ alone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.
Flower buds and immature embryos of P. nil Chois., which were grown in vivo , were material for the study. Flower bud (2-3 mm size) were treated with osmotic/trophic shock (12% sucrose) for 24h. After this time these explants were exposed on regeneration medium (MS supplemented with BAP in concentration 5 mg.dm-3 and NAA in concentration 0,1 mg.dm-3). After 4-6 weeks organogenesis of shoots was observed. Plantlets were isolated and transferred on MS medium with addition of GA3 [0,5 mg.dm-3] and NAA [0,1 mg.dm-3]. The plantlets developed into whole plants. Those plants were able to produce flower without photoperiodic induction, because these shoots regenerated from inducted tissue and ?remembered? this information. Immature embryos were isolated from previously sterilised fruit and afterwards transferred to MS without plant hormones. The embryos were cut across their axis. After 4-6 weeks of cultivation somatic embryos were formed in the injury place (hypocotyl region). These embryos were isolated and transferred on the same medium, which was used in shoots regenerated from flower buds. Embryos were converted into complete plants, but they weren?t able to flower without photoperiodic induction. However even the smallest embryos, which were used to our study (1 mm long) were able to flower, after a single induction cycle (16 hour of darkness), when the induction was given directly after isolation of embryos.
The aim of the performed investigations was to determine which part of the embryo has the optimal properties for callus induction at low concentrations of 2,4-D (2 mgdm-3). The explants were cut along or across the embryo axis into 2, 3, 4 fragments and cultured on the Murashige and Skoog medium for 6 weeks. It was found that callus derived from mature embryos (dry seeds) is characterised by comparable fresh and dry mass and by similar changes in size of the cells with fragments derived from mature embryos (but before the dormant) and from immature embryos. Essential difference was observed in the germination ability of the explants. The percentage of germination of mature embryo fragments was the lowest. It was demonstrated that despite of the embryo type, fragments originating from its central part appeared the most suitable for in vitro culture. In the case of these fragments the highest increase of the fresh mass of callus tissue, optimal morphological features and the appearance of meristematic centres were observed.
Chimera is a composite organism, consisting of cells derived from more than one embryo. The first experimental chimera was produced in 1961 and until today, chimeric animals have been widely used in mammalian experimental embryology. Numerous aggregation and injection techniques have been used to produce chimeras. Although most experimental chimeras were made of murine embryos, chimeric animals have been also produced in such species as: rat, hamster, deer mouse, rabbit, pig, sheep and in cattle. Some successful attempts to produce interspecific chimeras have been also made. Pigmentation is still widely used as a marker of chimerism, but new transgenic markers are now available. New methods emplying chimeric technique, like blastocyst complementation assay and lethal phenotypes rescue, provide new insights into developmental genetics. Nowadays, chimeric embryos are also likely to play a major role in the production of transgenic animals with the help of chimeric cloning technique.
Effects of fetal calf serum (FCS) or bovine serum albumin (BSA), with or without vitamin E (vit. E) or phenazine ethosulfate (PES) supplementation on developmental competence and quality of cultured porcine embryos were examined. The experiment was done on zygotes and 2-cell embryos obtained from superovulated gilts. Morphologically normal zygotes were cultured in vitro in NCSU-23 medium supplemented with: experiment 1-0.004 g/ml BSA, 10% FCS, protein-free (control); experiment 2-0 (control), 25, 50 or 100 M vit. E; experiment 3-0 (control), 0.025, 0.05 or 0.075 M PES. Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL staining. Presence of BSA in culture medium increased significantly morula and blastocysts production as compared to FCS (P<0.001) and protein-free group (P<0.05 and P<0.001, respectively). The blastocysts cultured in protein-free medium had higher average number of apoptotic nuclei and DNA fragmented nucleus index as compared to the BSA (P<0.05 and P<0.01, respectively) and FCS (P<0.5) group. Supplementation in culture medium of 100 M vit. E increased blastocyst production as compared to control and 50 M vit-E (P<0.05). Both the number of cells per and percentage of TUNEL positive nuclei per blastocyst were slightly lower in PES treated than control groups.
Prequisites for successful transformation are developed protocols on efficient regeneration of adventitious shoots or somatic embryos. The most important works concerning regeneration, the objectives and achievements in rose transformation are discussed.
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