We developed several novel tools to genome wide screen for CNVs and SNPs in single cells. When applied to cleavage stage embryos from young fertile couples we discovered, unexpectedly, an extremely high incidence of chromosomal instability, a hallmark of tumorigenesis (Vanneste et al., Nature Medicine, 2009; Vanneste et al., Hum.Reprod., 2011). Not only mosaicisms for whole chromosome aneuploidies and uniparental disomies but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres were detected. Based on the copy number changes that were observed in the blastomeres it was hypothesised that chromosome breakages and fusions occur frequently in cleavage stage human embryos and instigate subsequent breakage-fusion-bridge cycles and that the DNA breaks present in spermatozoa might trigger this CIN. To test these hypotheses, we genotyped both parents as well as 93 blastomeres from 24 IVF embryos and developed a novel SNP-array based algorithm to determine the parental origin of (aberrant) loci in single cells. Paternal as well as maternal alleles were commonly rearranged in the blastomeres indicating that sperm-specific DNA-breaks do not explain the majority of these structural variants. The parent-of-origin analyses together with microarray-guided FISH analyses demonstrate the presence of inv dup del chromosomes as well as more complex rearrangements (Voet et al., Hum.Mutation, 2011). These data provide unequivocal evidence for breakage-fusion-bridge cycles in those embryos and suggest that the human cleavage stage embryo is a major source of chromosomal disorders. The type of rearrangements observed can likely explain the majority of constitutional rearrangements seen in miscarriages as well as live births such as deletions, duplications, inverted deletions duplications, ring chromosomes and mosaicisms of all of those rearrangements. The high frequencies of chromosomal imbalances in cleavage stage embryos make it likely that a substantial number of chromosomal rearrangements originate post-zygoticaly.

DNA methylation is one of the important modifications in epigenetic gene regulation, and the control of DNA methylation status is a prerequisite for normal early EMBRYOGENESIS and cell differentiation. We have been analyzing two proteins, PGC7/Stella and MILI, which play crucial roles in the DNA methylation in early EMBRYOGENESIS and spermatogenesis, respectively.Although global demethylation occurs soon after fertilization, demethylation does not take place on the whole genome evenly. Maintenance of the methylation in the imprinted genes and epigenetic asymmetry between parental genomes, i.e., delayed demethylation of the maternal genome after fertilization are good examples of the protection of DNA methylation status. As one of the topics, I will show that PGC7/Stella, a maternal factor essential for early development, plays some roles in the protection of the DNA methylation in several genomic imprinting loci and epigenetic asymmetry. After determining that PGC7/ Stella binds to Ran binding protein 5 (RanBP5), a nuclear shuttle transporter protein, we examined the exact time when, and subcellular location where, PGC7/Stella functions, using mutants of PGC7/Stella and RanBP5. PGC7/ Stella turn out to be implicated in protecting the maternal genome from demethylation only after localization to the nucleus and maintaining the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is an indispensable methylation protector involved in epigenetic reprogramming after fertilization.The other topic I will introduce is the function of small RNA in gene silencing. Gene silencing in mammals is believed to be controlled by RNA interference in the same way as in other organisms; however, the molecules involved in the process remain unclear. The Argonaut proteins which include Piwi family proteins, are the candidates for controlling the silencing process. We have shown that a member of mouse Piwi family, MILI, plays crucial roles in the piRNA class of small RNA processing and/or protection, and subsequent gene silencing of retrotransposons through de novo DNA methylation at early spermatogenesis

Induction of somatic EMBRYOGENESIS was attempted in this study. Explants type (anthers, hypocotyls, cotyledons, leaf discs and petioles), media type (B5 and MS); auxins (NAA and 2,4-D) and cytokinins (TDZ, BAP) as well as environmental conditions (light and dark, temperature) were evaluated. Embryogenic callus of E. camaldulensis originating from hypocotyl explants was formed on the primary medium and developed on the secondary .medium' when specific hormonal formulation was used. A large number of synchronous embryos germinated and matured in the light. Without growth regulators. Secondary embryos were also obtained on solid Inedia. E. sargentii produced some asynchronous embryos. When the same method was used for somatic embry Digenesis of other Eucalyptus spp. rhizogenesis, or organogenesis, resulted, but no EMBRYOGENESIS. Attempts to produce embryogenic suspension cultures were unsuccessful. Plantlets produced from somatic embryos were successfully weaned and grown in the greenhouse for several months and their appearance was morphologically normal.

Regenerated plantlets were obtained from Ferula assa-foetida (Apiaceace) through indirect and direct somatic EMBRYOGENESIS, for the first time. Callus was induced on hypocotyl explants from seedlings of two ecotypes (Shirkooh and Tabas) on Murashige and Skoog (MS) basal medium supplemented with 0.5-4 mg/L kinetin along with 0.1-1 mg/L a-naphthalene acetic acid (NAA) for 12 weeks. Embryogenic calli developed within 4 weeks after transferring the calli to hormone-free MS medium. Induction/maintenance MS medium supplemented with 1.5 mg/L kinetin and 1 mg/L NAA was most effective and provided a high EMBRYOGENESIS frequency (31%) associated with a large mean number of mature somatic embryos per explant (8.4) in Tabas ecotype. According to our data, the presence of kinetin in the callus induction medium with NAA enhanced subsequent differentiation of somatic embryos on the hormonefree medium. About 40-50% of regenerated somatic embryos germinated into complete plantlets. Direct somatic EMBRYOGENESIS without an intervening callus phase was induced from intact seedlings on hormone-free medium within 12 weeks. Embryo induction was observed all over the seedling surface with the highest numbers on hypocotyls segments. By this procedure, the maximum mean number of embryo per seedling was 42 in Shirkooh ecotype and more than 50% of cotyledonary embryos were developed not only into normal plantlets, but rooted simultaneously when cultured on hormone-free MS medium. Also, histological observations revealed different stages of embryogenicity such as globular, heart, torpedo, and cotyledonary stages in F. assa foetida.

The aim of this study was production of haploid plants, using microspore culture method, in to be used in rapeseed breeding programs. Donor plants (cv. Global) were grown at a day/night temperature of 15/10˚C (16/8 hours). After 90 days microspores were isolated from buds 2.5-3.5 mm containing microspores at late-uninuclate to early-binucluate stages and cultured in a modified medium (NLN-13). Samples were incubated at 30˚C and in darkness for 14 days then transferred to a shaker at 25˚C for 20 days. In this experiment, the effect of different microspore densities included of: 60000, 40000 and 20000 microspores per ml, was used to study the EMBRYOGENESIS of microspores. Indeed, effect of embryo size (2-3, 3-4, 4-5, 5-6 and greater than 6 mm) on seedling germination properties like shoot formation, secondary EMBRYOGENESIS and root formation were also studied. Results showed significant differences between various culture densities (p<0/01). The density of 60000 microspores produced 1031 embryos in each petri dish (containing 12/5 ml culture medium) and was the best density in this study. The size of embryo showed significant effects on shoot and root formation at 1% of probability level. The size of embryo also showed significant differences on secondary EMBRYOGENESIS at 5%. Probably level in this expriment, it was observed that 73.33 % of embryos greater than 6 mm produced normal shoot had the best form of germination. In this study 43% of embryos were also regenerated.

Light spectrum is one of the environmental cues that influence plant growth and development. Light is a stimulating factor for induction of somatic embryos during tissue culture practices. To accelerate the direct EMBRYOGENESIS, six different light spectra including: white (W), red (R), blue (B), green (G), red + blue (R+B) and red + far red (R+FR) together with dark condition (D), in combination with thidiazuron (TDZ) in four concentrations (0, 0. 5, 1. 5 and 3 mg L-1) were used. Inter-simple sequence repeat was used for identification and genetic stability analysis of somatic regenerated plantlets. Intact protocorm explants showed higher potential for direct somatic EMBRYOGENESIS (DSE) than the other explants. The rate of DSE was highly dependent on the concentration of TDZ and its interaction with light spectra. R and R + FR spectra with 3 mg L-1 TDZ on intact protocorms and R+FR with 3 mg L-1 TDZ were efficient treatments to induce DSE without somaclonal variation. G light spectrum has also significant effects on DSE of protocorm explants. The amplified products showed 26 scorable bands and regenerates were completely identical to the mother plant. In conclusion, this protocol provides way to regenerate plants through EMBRYOGENESIS, and is a reliable protocol to obtain proper development and genetic stable Phalaenopsis embryos.