Le sujet de ma thèse était de décrire sur le plan moléculaire le processus d'embryogenèse somatique chez la vigne. Pour cela, les étapes-clés d'entrée et de sortie du cycle d'embryogenèse secondaire ont été caractérisées par l'analyse de l'expression de quelques gènes impliqués dans le développement ou la défense, en particulier les gènes PR10.Grâce à l'exploitation de la séquence complète du génome de Vitis vinifèra disponible sur le site du Genoscope, j'ai pu caractériser exhaustivement la famille multigènique des PR10. Celle-ci est composée de 17 séquences disposées en tandem et formant un cluster compact sur le chromosome 5, dont 3 pseudogènes et au moins 13 séquences transcrites. L'expression de 10 de ces gènes a d'abord été analysée par RT-PCR semi-quantitative dans différents organes de la plante et dans des tissus traités au 2,4-D. Elle suggère une diversification fonctionnelle marquée. De plus, le niveau d'expression de plusieurs gènes PR10 est élevé dans les cals embryogènes, suggérant qu'ils pourraient jouer un rôle lors de l'embryogenèse somatique. L'étude de l'expression des gènes PR10 par RT-PCR quantitative en temps réel dans différents tissus ayant montré une capacité embryogénique variable lorsqu'ils sont soumis à un traitement par le 2,4-D met en évidence que le niveau d'expression varie entre les gènes et selon les tissus. L'expression de certains gènes est fortement induite par le 2,4-D dans les tissus à capacité embryogénique et seulement faiblement dans les tissus ne donnant jamais d'embryons somatiques, ce qui suggère fortement que ceux-ci pourraient être des marqueurs de la capacité embryogénique chez la vigne. / The objective of my work was to analyse the somatic embryogenesis process of Vitis vinifera at a molecular scale. Thus, the expression of genes implied in development or defence, especially PR10 genes, was monitored during the key-steps of entrance and exit of secondary somatic embryogenesis. The complete sequence of the Vitis vinifera genome available on the Genoscope website allowed the exhaustive characterization of the PR10 multigene family, which is constituted by 17 sequences localised on a tandem array on the chromosome 5. Among these 17 sequences, 3 are pseudogenesand at !east 13 are transcribed sequences. The expression of 10 PR10 genes was first monitored in various grapevine tissues and in tissues after 2,4-D treatment using semi-quantitative RT-PCR. The results suggest a strong functional diversification. Moreover, the expression of several PR10 genes is high in embryogenic calli, suggesting that these genes could intervene in somatic embryogenesis. The expression of PR10 genes was also monitored in tissues showing different somatic embryogenic capabilities under 2,4-D treatment using quantitative RT-PCR. The results show that regulation of PR10 genes is dependent of the gene and tissue considered. Moreover, the expression of some genes is highly induced by 2,4-D treatment in tissues having embryogenic capability, white it is only weakly induced in tissues having no embryogenic capability, suggesting that these gene could be markers of embryogenic capability in grapevine.

No description available.

Cloning of trees using somatic embryogenesis could have a major impact on tree breeding and commercial plantation forestry. To initiate somatic embryogenesis in Anatolian black pine (Pinus nigra Arnold. subspecies pallasiana), one-year old cones containing immature seeds were collected from eight trees located in METU campus, Ankara. Embryogenic tissues were derived from immature zygotic embryos excised from the seeds. The zygotic embryos at the time of collection were at the precotyledonary stage of development. For this study, Douglas-fir cotyledon revised medium (DCR) supplemented with 13.6&micro / M 2,4-D, 2.2&micro / M BAP, 0.5 g/L casein hydrolysate, 0.25 g/L L-glutamine and 3% sucrose was used. The media was solidified with 0.2% gelrite. Embryogenic tissue initiation was calculated for each genotype and collection date. Overall initiation frequencies were recorded as 0.92% for 2004 and 1.96% for 2005. Highest initiation frequency was calculated for 5-July 2005 sampling time (4.06). ANOVA revealed significant differences between trees and collection date for initiation frequencies. Also, ECL (Established cell lines) recorded after five subcultures. Overall, 0.38% and 0.62% of the initial explants were converted into ECLs for 2004 and 2005 respectively.

Calcium ion plays a pivotal role as second messenger during signal/response coupling in plant cells (Trewavas, 1999). Elevations of cytosolic Ca2+ occur in plants as a consequence of abiotic and biotic stresses, environmental and hormonal stimuli. However, the molecular mechanism by which changes in cytosolic calcium are sensed and transduced in the plant cell has not been completely elucidated. The detection of Ca2+-binding proteins, especially Ca2+-dependent protein kinases (CDPKs) in plants led to drawing analogy with animal systems wherein the Ca2+-message is perceived and transduced by proteins that bind Ca2+. CDPKs are stimulated by the direct binding of Ca2+ to their endogenous calmodulin (CaM) -like domain (Harper et al, 1991). CDPKs exist as multiple isoforms in a single species, and show tissue-specific and developmentally regulated expression. Furthermore, the diversity among different CDPK isoforms with respect to Ca2+-binding properties, activation, substrate specificity, regulatory mechanisms and other kinetic properties suggest their specialization in the regulation of distinct signaling pathways. These observations therefore have led to the speculation that most of the Ca2+-mediated signal transduction in plants occurs via the mediation of CDPKs (Harmon et al, 2000). Over the last 15 years there has been a dramatic unfolding of information on Ca2+-mediated signaling in plants. Nevertheless, little is known about the environmental/hormonal signals and the signaling events that regulate early plant developmental processes such as embryogenesis, seed development and germination. The present investigation was initiated with the objectives 1) to determine the role of Ca2+ during embryogenesis, 2) to examine the involvement of a CDPK during early developmental processes in sandalwood plant (Santalum album L.) and 3) to purify and biochemically characterize this CDPK. The study initially investigated the possible involvement of calcium-mediated signaling in the induction/regulation of somatic embryogenesis from proembryogenic cells of sandalwood. 45 Ca + uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of proembryogenic cells in response to culture conditions conducive for embryo development. Sandalwood proembryogenic cell masses (PEMs) were obtained in the callus proliferation medium that contains the auxin 2,4-D. Subculture of PEMs into the embryo differentiation medium which lacks 2,4-D and has higher osmoticum resulted in a 4-fold higher 45Ca2+ incorporation into the symplast. Fura-2 based ratiometric analysis also showed a 10-16- fold increase in the [Ca2+]cyt of PEMs under identical culture conditions, increasing from a resting concentration of 30-50 nM to 650-800 nM. Chelation of exogenous Ca2+ with EGTA arrested such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrested embryo development and inhibited the accumulation of a Ca2+-dependent protein kinase (swCDPK) in embryogenic cultures. However, such culture conditions did not cause cell death as the PEMs continued to proliferate to form larger cell clumps. Culture treatment with W7 reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving swCDPK and/or CaM caused inhibition of embryogenesis. These observations suggest a second messenger role for exogenous Ca2+ and the existence of Ca2+-mediated signaling pathway(s) during sandalwood somatic embryogenesis. The detection of a 55 kD protein showing cross reactivity with polyclonal antisoybean CDPK and the detection of Ca2+-dependent protein kinase activity in protein extracts from somatic embryos, prompted investigation on the spatio-temporal accumulation and activity of a CDPK in different developmental stages of sandalwood. Western blot analysis and protein kinase assays identified a Ca2+-dependent protein kinase (swCDPK) of 55 kD in soluble protein extracts of different developmental stages of sandalwood somatic embryos. However, swCDPK was not detected in plantlets regenerated from somatic embryos. swCDPK exhibited differential expression and activity in the developmental stages of sandalwood. Zygotic embryos, endosperm and seedlings showed high accumulation of swCDPK. However, the enzyme was not detected in the soluble proteins of shoots and flowers of sandalwood tree. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages, the enzyme being localized strongly in the storage bodies of the endosperm cells. Interestingly, these storage bodies were thereafter identified as oil bodies, suggesting that a Ca2+-mediated regulation of oil hydrolysis and/or mobilization might be operative during seed germination. swCDPK in the zygotic embryo was found to be inactive during seed dormancy and early stages of germination, indicating a possible post-translational hibition/inactivation of the enzyme during these stages. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation and germination thus suggests involvement of the enzyme in these early developmental processes. In view of the diversity exhibited by members of the CDPK family, characterization of swCDPK, the early development specific CDPK from sandalwood was undertaken. Purification of swCDPK was achieved by chromatography on DEAE-cellulose, hydroxyapatite and Blue-Sepharose. The purified enzyme resolved into a single band on 10 % polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK was strictly dependent on Ca2+, K0.5 (apparent binding constant) for Ca2+-activation of substrate phosphorylation activity being 0.7 μM and for autophosphorylation activity —50 nM. Ca2+-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC50 for W7 = 6 μM, for W5 = 46 μM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a Km value of 13 mg/mL for histone III-S and a Vmax of 0.1 nmolmin-1rng-1. The enzyme exhibited high specificity for ATP with a Km value of 10 nM. Titration with Ca2+ resulted in enhancement of the intrinsic emission fluorescence of swCDPK and a shift in the λmax emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca2+-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca2+ levels required for autophosphorylation and substrate phosphorylation help elucidate the possible mode of Ca2+ activation of this enzyme.

The mammalian embryo is encased in a glycoproteinaceous covering, the zona pellucida (ZP/zona) during preimplantation development. Prior to implantation into the recipient maternal endometrium, the blastocyst has to hatch out of this zona. This is a critical and an important event for the successful establishment of pregnancy. Hatching in mammals is characterised by the expansion of the blastocyst, followed by the nicking of the zona and extrusion of the blastocyst by repeated contraction-expansion cycles, thereby leaving the empty zona behind. In species such as the mouse, cow and primates, the empty zona is left behind in the uterine lumen. However, in the hamsters, the features associated with hatching are characteristic for this species. Firstly, the blastocyst remains predominantly in a deflated state. Secondly, the zona undergoes focal rupture which is followed by the complete dissolution of the zona. Third, trophectodermal projections (TEPs) present in the blastocysts, aid the hatching of the blastocyst. Hence, this study was aimed to identify the molecular players involved in hamster blastocyst hatching and to study their embryo-endometrial expression. Earlier work in the laboratory has demonstrated the involvement of cysteine protease-like factors in hamster blastocyst hatching (Mishra and Seshagiri, 2000a). Broad spectrum cysteine protease inhibitors, E-64 and PHMB, completely blocked the hatching of blastocysts. To identify the class of cysteine proteases involved in this phenomenon, class-specific inhibitors were used in this study. Calpain and caspase inhibitors, calpastatin and Z-VAD- FMK, respectively, did not block hamster blastocyst hatching (Fig 2.2). Cathepsin (cts)-specific inhibitors, cystatin-C and peptidyl diazomethane (PPDM) blocked hatching of embryos in a dose-, time- and embryo-stage dependant manner (Figs 2.5, 2.6, 2.10 and 2.11). Continuous exposure of 1.0 µM cystatin to expanded or deflated blastocysts completely blocked hatching (Fig 2.3Aii, iii), without effecting their viability (Fig 2.3Bii and iii). Deflated blastocysts exposed transiently to 1.0 µM cystatin, for 12 or 6 h failed to hatch, but could overcome the inhibition and exhibited hatching, when transferred to fresh, inhibitor-free medium (Fig 2.6). Effect of the inhibitor was less pronounced in the deflated blastocysts when compared to expanded blastocysts (Figs 2.5 and 2.6). The viability of the cystatin-treated embryos was not affected as assessed by vital-dye staining and also their ability to attach and exhibit trophoblast (TB) proliferation on serum-coated dishes was not compromised. The area of the TB outgrowth of cystatin-treated embryos was similar to that of the untreated embryos (Fig 2.9). The inhibitory effect of PPDM, an irreversible inhibitor of cts, on blastocyst hatching was demonstrated. Expanded and deflated blastocysts exhibited a dose-dependent inhibition in hatching, following the exposure to 0.5 and 1.0 µM of PPDM (Figs 2.10A and 2.11A). When treated with the inhibitor for 6 or 12 h, there was a transient inhibition in hatching, as blastocysts could overcome the inhibition and exhibit hatching following transfer to inhibitor-free, fresh medium. Inhibitor-treated hatched blastocysts, when transferred to serum-coated dishes, attached and exhibited TB outgrowth, similar to untreated embryos (Figs 2.13 and 2.14). A PPDM-interacting protease was localised to the cytoplasm of the embryonic cells in the hamster blastocyst, suggesting that the embryo is the source of the zona lysin. Two forms of the enzyme, a probable variant zymogen of molecular mass 65 k and an active form of molecular mass 32 k were detected in the blastocysts (Fig 2.15). In vitro susceptibility of hamster zona to cathepsins is significantly different from that of other species zonae such as the mouse, rat, monkey and human zonae (Table 2.2). All these lines of evidence unequivocally demonstrate the involvement of cathepsins in hamster blastocyst hatching, which is in sharp contrast to what is observed in the mouse, where serine proteases such as strypsin/hepsin, ISP-1 and -2 are reported to play an important role in blastocyst hatching. However, since extensive inhibitor studies were not performed using embryos from other species, it is possible that cysteine proteases maybe involved in the hatching of blastocysts from other species. Having shown the role of cathepsins in hamster zona dissolution, expression of the cathepsins in preimplantation embryos was investigated. Hamster specific cts–L, -B and –P were amplified from day 14 placenta using mouse primers and the amplicons were found to be highly homologous to the cts of other species (Fig 3.2). Hamster and mouse preimplantation embryos i.e., 8-cell, morula and blastocyst were found to express cts–L, -B and –P transcripts (Figs 3.6 and 3.10). Cts-P, present only in the TBs of the placenta (Fig 3.4), for the first time, was also shown to be present in the preimplantation embryos. The immunoreactive cts-L and -P proteins were detected in blastomeres of 8-cell embryo, in the inner cell mass (ICM) and trophectoderm (TE) of the blastocyst (Figs 3.7 and 3.8). These cathepsins could probably correspond to the PPDM- interacting enzymes of molecular mass 32 and 65 kDa, described above. (fig) Fig 5.1. Overview of the expression and the role of embryo-endometrial cathepsins in blastocyst hatching in the golden hamster. Cathepsins ( ) produced by the inner cell mass ( ) or the trophectoderm ( ) of the blastocyst or the endometrial cells ( ) act on the zona matrix ( ), bringing about its lysis. The cathepsins are secreted into the peri-vitelline space or are carried by trophectodermal projections (TEPs, yellow projections) to the zona. Also shown are endogenous inhibitors and growth factors that can regulate these cathepsins. A striking observation made in this study was the detection of the immunoreactive signals for cathepsins in the zona matrix of blastocysts. Since hamster blastocysts possess extracellular projections (TEPs), it is possible for these projections to participate in the transport of cathepsins from TE cells to the zona; as the localisation of the proteases to these projections was demonstrated (Fig 3.9). Also, since the actin-based projections are highly undulating structures, they might potentiate the mechanical rupture of the zona during hatching, apart from acting as carriers for the proteases. Hence, during hatching of the hamster blastocyst, cathepsins, expressed in the ICM and the TE, might be secreted transiently into the peri-vitelline space, whereby they can act on the ZP. Alternatively, in the absence of any apoptotic cells in the embryo that can release the cell contents (Fig 3.13), the cathepsins may be deposited by TEPs in specific pockets of the zona matrix, thereby causing focal zona lysis. In vivo, the hatching of the blastocysts is brought about by both embryonic and maternal proteases. Cts–L and -B transcripts were detected in the maternal endometrium during different stages of the reproductive cycle and early pregnancy (Fig 4.1 and 4.3). Immunoreactive cts-L protein was detected in the uterine luminal epithelium and the stromal cells (Fig 4.5). In the uterus, the PPDM-interacting 32 kDa form was in abundance compared to the 65 kDa form (Fig 4.6). Hence, uterine cathepsins might play a major role in the remodelling of the extracellular matrix during estrous cycle and pregnancy. However, the role of these cathepsins in causing zona dissolution during blastocyst hatching, along with embryonic proteases cannot be ruled out. Reports of recurrent miscarriages in women with low serum cystatin levels imply a role for cysteine proteases in early pregnancy events like blastocyst development, hatching and implantation. Hence, these studies, described in the thesis, could form a basis to investigate the role of cathepsins in early human development. Taken together, the results demonstrate the involvement of embryo-derived cathepsins in hamster blastocyst hatching. These cathepsins may be secreted into the peri-vitelline space or transported to the zona matrix by TEPs (Fig 5.1). Additionally, in vivo, endometrial cathepsins might aid the embryonic zona lysins in the complete zona dissolution. The regulation of these proteases by growth factors, cytokines and their specific inhibitors needs to be explored. (For figure pl see the original document)

The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize several candidate genes within two known regions of genomic instability on chromosome 18 and chromosome 4. This fish that were used in this study had been previously classified as genomic instability (gin) mutants due to increased incidence of somatic mutation during the early stages of embryogenesis, that can be detected with the mosaic eye assay at 48-72 hpf. Using published genome and mapping data, several candidate genes for two of the gin mutations were identified and studied during early zebrafish development. The gin mutations are heritable, ENU-induced, and have both maternal and zygotic effects during zebrafish development. The first aim of this project was to study the normal gene characteristics of the gin-10 candidate genes, synbl, rfx4, and sir2 that are located on chromosome 18. Semi-quantitative RT-PCR, whole-mount in situ hybridization, and gene knockdown (using morpholino oligonucleotides) techniques were utilized in both wildtype and transgenic (Tg-synbl) zebrafish lines to gain an understanding of the function of each of these genes during zebrafish embryogenesis. Additionally, the synbl paralog, ric8a, was also explored, as it has been implicated in the control of asymmetric cell division in C. elegans. Single gene knockdowns were performed for each candidate in the golden heterozygous (pigment mutant) zebrafish background to test for genomic instability activity. Genomic instability activity was not observed, however the results showed that these genes are expressed throughout zebrafish embryogenesis, and are necessary for the proper development of the central nervous system, notochord and tail, as well as metabolic functions in the early embryo. Moreover, the transgenic line used for the paralog studies of synbl and ric8a was incorrectly genotyped. Using PCR analysis and sequencing, it was found that the viral insert for the Tg-synbl fish was disrupting the cry1b gene on an adjacent contig. The second aim focused on the gin-12 region on chromosome 4, where the mdm1 gene is located. Originally cloned from a transformed mouse cell line with mdm2, the function of the mdm1 gene in these cells or during development had not yet been identified. To allow the Mdm1 protein to be evaluated, custom antibodies targeting Mdm1 were produced and the detection of Mdm1 optimized in zebrafish embryos. This would allow us to then determine whether Mdm1 was a possible regulator of the p53-Mdm2/Mdm4 pathway. Additionally, the mdm1 gene was studied in situ and in vivo to determine the normal gene expression patterns and developmental role in the embryonic zebrafish. Moreover, this gene was also studied in the golden heterozygous zebrafish line to assess whether it had a role in modulating genomic instability activity using the mosaic eye assay. Collectively, morpholino oligonucleotides, RNA rescue, whole-mount antibody staining, and overexpression studies suggest that the mdm1 gene is involved in the development of the eye and portions of the central nervous system, but did not appear to be the gin-12 mutant. While the genes in this study did not appear to have genomic instability activity in the embryonic zebrafish based on the mosaic eye assay in the golden heterozygotes, normal developmental gene expression patterns were identified for synbl, ric8a, rfx4, sir2, and mdm1 in wildtype zebrafish embryos. Additional information was gained by the reverse genetic studies using gene knockdowns, which identified the functional roles of these genes at various stages of embryogenesis. Notably, it was determined that the mdm1 gene may be involved in retinal degenerative diseases based on our studies and recently published data. Future research of the Mdm1 protein should identify protein interactions and the specific role during eye development and retinal diseases.

Bei der routinemäßigen Anwendung der Mikrosporenkultur zur Herstellung doppelt-haploider Linien kommt es bis heute zu Engpässen in der praktischen Rapszüchtung. Die Hauptprobleme stellen eine unzureichende Colchizin-induzierte Diploidisierungsrate und eine niedrige direkte Regeneration von Pflanzen aus Mikrosporen-Embryonen dar. Ein hoher Prozentsatz an Rapsembryonen aus Mikrosporenkultur durchläuft den Prozess der sekundären Embryogenese, der eine zeitintensive Subkultivierung erfordert. Hierbei werden die direkten Sprossansätze wiederholt von undifferenziertem Gewebe freigeschnitten bis eine Überführung in Erde und die letztendliche Regeneration zu doppelt-haploiden Pflanzen möglich ist. Die vorliegende Doktorarbeit besteht aus zwei Studien, die sich mit dem Thema: „Methodische Verbesserungen der Mikrosporenkultur in Brassica napus L.“ auseinandersetzen. Ziel der ersten Studie war die Erhöhung der Colchizin-induzierten Diploidisierungsrate von Mikrosporen ohne die Regeneration von Pflanzen aus den Mikrosporen-Embryonen zu verringern und damit die Entwicklung zu doppelt-haploiden Pflanzen zu verzögern. Aufgrund der hohen Toxizität von Colchizin wurden die weniger toxischen Mitosehemmstoffe Amiprophos-methyl (APM) und Pronamid, die eine höhere Affinität zu Pflanzentubulin als Colchizin besitzen, allein und in Kombination mit Colchizin untersucht. Eine Kombination dieser Mitosehemmstoffe führte zu keiner effizienten Diploidisierungsrate; demnach konnte ein synergistischer Effekt ausgeschlossen werden. Die acht untersuchten Winterrapsgenotypen erzielten eine Diploidisierungsrate von 40% bis 64%. Die Mitosehemmstoff-Behandlungen der isolierten Mikrosporen variierten hierbei zwischen 33% (3 µM APM, 72 Stunden) und 70% (25 µM Colchizin, 72 Stunden). Ein signifikanter Einfluss der Mitosehemmstoffe auf die Konversionsrate der Mikrosporen-Embryonen konnte nicht nachgewiesen werden. In Abhängigkeit der Genotypen konvertierten 14% bis 23% direkt. Unterschiedliche getestete Colchizinkonzentrationen (250, 150, 125, 25 µM) zeigten für 4 untersuchte Genotypen eine Colchizin-induzierte Diploidisierungsrate von 58% bis 66%, wobei die Behandlung 250 µM Colchizin für 48h die höchste Rate aufwies. Ein signifikanter Einfluss von Dimethylsulfoxid (DMSO), das oftmals als Lösungsmittel der angewendeten Mitosehemmstoffe verwendet wird, konnte jedoch nicht in den untersuchten Konzentrationen (0,3% und 3%) in Kombination mit der Colchizin-Behandlung (250 µM, 72 Stunden) auf die Diploidisierungsrate und die direkte Konversionsrate nachgewiesen werden. Weiterhin wurden 17 Winterrapsgenotypen bezüglich ihrer spontanen und ihrer Colchizin-induzierten Diploidisierungsrate untersucht und deren Konversionsrate der Mikrosporen-Embryonen zu Regeneraten mit direkten Sprossansätzen bestimmt. Die ausgewählten Genotypen enthielten sowohl Sorten als auch F1–Hybriden. Die spontan-induzierte Diploidisierungsrate zeigte eine große Variation von 15% bis 69%. Im Vergleich dazu erreichte die Colchizin-induzierte Diploidisierungsrate Werte von 40% bis 83%. Die Mikrosporen-Embryonen der getesteten Genotypen wiesen ebenfalls eine große Spannbreite bezüglich ihrer direkten Konversionsrate auf. Die Ergebnisse zeigten keinen signifikanten Einfluss der Mitosehemmstoff-Behandlung auf den Regenerationserfolg der Mikrosporen-Embryonen zu Pflanzen. Sowohl die beobachtete spontane und die Mitosehemmstoff-induzierte Diploidisierung als auch die Konversionsrate der Mikrosporen-Embryonen zu Regeneraten mit direkten Sprossansätzen waren stark Genotyp-abhängig. Ziel der zweiten Studie war die Erhöhung der direkten Regeneration der Mikrosporen-Embryonen zu Pflanzen trotz der starken Abhängigkeit der Genotypen. Zunächst wurde der Einfluss von zehn unterschiedlichen Sprossregenerationsmedien mit und ohne Phytohormone (Gibberellinsäure, 6-Benzylaminopurin, 3-Indolylbuttersäure) und eine 14-tägige Kältebehandlung bei 4 °C (Lichtthermostat) auf die direkte Konversionsrate der Mikrosporen-Embryonen von 5 Winterrapsgenotypen untersucht. Die 14-tägige Kältebehandlung erfolgte sowohl unter acht Stunden Licht als auch in Dunkelheit. Die Standardkultivierung der Mikrosporen-Embryonen erfolgte im Kulturraum bei 26 °C und 12 Stunden Licht. 13% bis 39% der Mikrosporen-Embryonen konvertierten direkt, wobei die höchste Rate von 43% nach Kultivierung der Embryonen auf Gamborg B5-Medium mit 0.1 mg/L Gibberellinsäure resultierte. Die Mittelwerte der Konversionsrate der Mikrosporen-Embryonen zu Regeneraten mit direkten Sprossansätzen aller untersuchten Genotypen und Kulturmedien wurden durch die 14-tägige Kältebehandlung (28%) gegenüber der Standardkultivierung (14%) signifikant erhöht. Nachfolgend wurde der Einfluss der vier effizientesten Sprossregenerationsmedien und eine 14-tägige Kältebehandlung bei 1.5 °C und bei 4 °C (Lichtthermostat) auf die Konversionsrate von Mikrosporen-Embryonen von 13 Winterrapsgenotypen untersucht. Die Kältebehandlung bei 1.5 °C erfolgte unter Lichtabwesenheit als auch unter acht Stunden Licht. Die Kältebehandlung bei 4 °C erfolgte dagegen in Dauerlicht und Dauerdunkel. Zwischen 29% und 76% der Mikrosporen-Embryonen konvertierten direkt. Im Vergleich zur Kultivierung unter Standardbedingungen konnte mit der Kältebehandlung eine signifikante Erhöhung erzielt werden (von 21% auf bis zu 71%). Nach vorheriger Kultivierung der Mikrosporen-Embryonen auf den unterschiedlichen Kulturmedien variierte die Konversionsrate zwischen 50% (MS) und 60% (B5 mit 0.1 mg/L Gibberellinsäure). Die Ergebnisse der Untersuchungen zeigten, dass trotz einer vorherrschenden starken Abhängigkeit vom Genotyp, die direkte Konversionsrate der Mikrosporen-Embryonen mit Kältebehandlung (1.5 °C im Dauerdunkel) signifikant erhöht werden konnte. Fast alle Genotypen zeigten Konversionsraten der Mikrosporen-Embryonen von über 70%. Es ist demnach möglich die sekundäre Embryogenese und die damit verbundene zeitintensive in vitro-Subkultivierung erheblich zu reduzieren, und dadurch den Entwicklungsprozess von doppelt-haploiden Linien zur Verwendung in der praktischen Rapszüchtung zu beschleunigen.

Tyrimai atlikti 2005-2007m. LŽŪU Genetikos biotechnologijos laboratorijoje. Tirti veiksniai, įtakojantys vasarinių rapsų (Brassica napus var. oleifera) dvigubų haploidų linijų NL-302-01, NL-302-02, NL-302-25 somatinės embriogenezės procesą nesubrendusių zigotinių gemalų kultūroje. Tirtų genotipų eksplantai (zigotniai gemalai) kultivuoti maitinamosiose terpėse, besiskiriančiose terpės rūgštingumu. Eksplantai iš donorinių augalų tirti 14-29 amžiaus dienų po apdulkinimo. Nustatyta, kad vasarinių rapsų somatinės embriogenezės potencialas priklauso ne tik nuo nesubrendusio zigotinio gemalo amžiaus po apdulkinimo, bet ir nuo eksplanto genotipo ir terpės rūgštingumo. Daugeliu atveju didžiausią įtaką indukcijos intensyvumui turėjo genotipas. Didžiausiomis pirminės somatinės embriogenezės galimybėmis pasižymėjo linijos NL-302-25 gemalai 20-21 dienos amžiaus. Antrinės somatinės embriogenezės metu nustatyta, kad indukcijos potencialas buvo didesnis nei pirminės somatinės embriogenezės metu. Ekspalnto amžius įtakos soamtinių embrioidų formavimo kiekiu neturėjo. / The research was carried out in 2005-2007 in the laboratory of genetics and biotechnology of Lithuanian university of Agriculture Factors influencing double haploid lines’ NL-302-01, NL-302-02, NL-302-25 somatic embryogenesis in the culture of premature zygotic germs of summer rape (Brassica napus var. oleifera) were studied. The explants of the studied genotypes (premature germs) were cultivated in the nutritional environments differencing in pH. The explants from the donor plants were taken for the period of 14-29 days. It was determined that the potential of the somatic embryogenesis of the summer rape depends on not only on the age of the premature zygotic germ after the pollination, but also on the genotype and medium pH of the explants. In most cases the biggest effect on the intensity of the induction was caused by genotype. Line NL-302-25 cultivated 20-21 days after the pollination had the biggest potential of primary somatic embryogenesis. During the secondary somatic embryogenesis it was determined that the potential of induction was greater than during the primary somatic embryogenesis. The age of the explant had no effect on the quantity of formation of somatic embryos.

Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit, that are expressed on the surface of all metazoan cells. These bidirectional signaling molecules are involved in many well-known aspects of cell function, although the role of integrins in early embryonic development remains a mystery. The purpose of this study was to characterize S. purpuratus integrins and determine if they are necessary for early embryonic development. Full length cDNA sequences for four incomplete gene predictions, αC, αD, αF, and βD, were determined by amplifying overlapping fragments and sequencing EST clones. Each cDNA has a single open reading frame predicting a protein with canonical integrin features. QPCR results show αC, αD, and βD are expressed in the embryo at relatively constant levels during the first 96 hours of development. αF is expressed in blastulae, during morphogenesis and tissue differentiation, at up to 35 times the levels of mRNA in the egg. Using a morpholino antisense oligonucleotide to block translation of αC results in a higher than normal mortality rate (57.1%) by 24 hours of development and 36.7% of embryos during this period have defects in aspects of cell division. These results indicate that αC is an essential gene for early development and that it may function in coordination of mitosis and cytokinesis. The expression of multiple subunits and the demonstration that αC has an essential role suggests that there are several non-overlapping functions for integrins in early embryonic development.

In order to elucidate the molecular and biochemical events occurring in embryogenesis in Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco), an essential gene expressed highly during early embryogenesis was identified, cloned and further characterized in this study. Douglas-fir LYR/LVR eDNA was obtained using RT-PCR with specific primers. followed by cloning and sequencing. Northern blot analysis showed higher amounts of LYR/LVR transcripts in early-cotyledonary embryonic stages and megagametophytes when compared with mid- and late-cotyledonary embryos. LYR/LVR transcript levels declined in seeds (mature embryos) and seedlings. Differential regulation of LYR/LVR gene expression with response to brassinosteroid treatment of Douglas-fir seeds was studied. LYR/LVR mRNA showed higher accumulation in seeds treated with different concentrations of brassinosteroids. Bioinformatic analysis showed that Douglas-fir LYR/LVR protein may be an essential inner mitochondrial protein, NADH oxidoreductase necessary for energy production. The phylogenetic tree analysis was used to investigate the evolutionary relationship of the newly identified Douglas-fir LYR/LVR protein with closely related proteins (LYR family) in different organisms. InterPro, UniProt and Pfam results showed the sequence similarity of Douglas-fir LYR/LVR protein with other related members in Arabidopsis thaliana and Oryza sativa, indicating that the LYR complex contains short stretches of closely related proteins that are essential for energy production. Amino acids 19-90 in the LYR/LVR protein were highly conserved and is likely the functional LYR motif necessary for oxidoreductase activity.