Haploid Condition Research Articles

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.

Increased production of azadirachtin from an improved method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss.

This report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica, a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium with BAP (5 µM), 2,4-D (1 µM) and NAA (1 µM) containing 12% sucrose. The calli, induced, were further multiplied on 2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 µM) + NAA (0.05 µM) medium, were elongated on MS + BAP (0.5 µM) and multiplied on MS + BAP (1 µM) + CH (250 mg/l). Thereafter, the shoots were rooted on 1/4 MS + IBA (0.5 µM). Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as 2n=x=12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and mass spectroscopy. Maximum azadirachtin (728.41 µg/g DW) was detected in calli supporting best shoot proliferation while least (49 µg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous controlled production of high value compounds, round the year, independent of geographical and climatic barrier.

63 OOCYTE GENOME CLONING USED IN TRANSGENIC BOVINE EMBRYO PRODUCTION

Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogenetic haploid blastomeres (PHB) from these embryos should be considered as a clone of the original gamete. Our main objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Moreover, we generated biparental homogeneous transgene-expressing embryos using PHB that express a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation in 5 μM ionomycin for 4 min followed by culture in SOF for 3 h to permit second polar body extrusion and subsequently treated with 1.9 mM DMAP for 3 h. To generate transgene-expressing PHB, parthenogenetic embryos were injected 3 h post-activation with pCX-EGFP–liposome complexes. These treatments were analysed by Fisher test (P < 0.05). The cleavage rate of the haploid and diploid parthenogenetic embryos control was 87.3% (103/118) and 88.5% (108/122) respectively; (P > 0.05). When the haploid activated oocytes were injected with pCX-EGFP–liposome complexes, the cleavage rate was 84.4% (54/64). These embryos showed 54.7% (35/54) of EGFP expression. In the second experiment, haploid parthenogenetic embryos (4 to 16 cells) were disaggregated and the PHB obtained were fused with a zona-free haploid male hemizygotes in order to reconstruct biparental embryos. Haploid male hemizygotes were generated by fertilization of enucleated oocytes. The haploid condition of parthenogenetic embryos and male hemizygotes was confirmed by karyotype analyzes. Reconstructed embryos were cultured in SOF medium in well of well system in 5% O2, 5% CO2, and 90% N2 at 39°C. The development of the reconstructed embryos expressing or not expressing EGFP reached 78.4% (29/37) and 61.1% (80/131) of cleavage; and 10.8% (4/37) and 8.4% (11/131) of blastocysts, respectively; (P > 0.05). EGFP expression was observed in 100% of the reconstructed embryos, with 96.6% (28/29) of homogenic expression. The biparental IVF control showed 64.4% of cleavage and 22.5% of blastocysts (n = 98). The mean cell number of blastocysts were significantly different among the reconstructed biparental embryos (mean = 72.8) and the biparental IVF-embryos control (mean = 97.0). Additionally, the Oct-4 pattern expression was examined in the blastocysts, by inmunocytochemistry. Blastocysts displayed a pattern of Oct-4 expression similar to IVF-blastocyst control. Finally, biparental reconstructed blastocysts (n = 2) were transferred in recipient cows obtaining 1 pregnancy detected at 2 months after embryo transfer. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that it is an efficient technique to generate homogeneous transgene-expressing embryos. Haploid oocyte genome replication would have high potential in livestock production, as it increases the number of embryos produced from a single oocyte, and allows the selection of favourable genetic characteristics before biparental embryo reconstruction.

AZFc region of the Y chromosome shows singular structural organization

Owing to clonal inheritance, haploid status and lack of recombination, structural polymorphism in the human Y chromosome is more prevalent than that in the remaining parts of the genome. We studied structural organization of the AZFc region, assessed microdeletions therein and studied copy number variation (CNV) of several candidate genes in 750 Indian males. FISH mapping of 13 Y-specific BAC/cosmid clones uncovered a hitherto unreported AZFc configuration showing inter-DAZ gene sequence onto the Yp instead of Yq region. Such inter-DAZ gene arrangements were also detected in five German males (European Y). In 40-50% males, partial u3 and one of the green amplicons, g1, g2 or g3 was present on the Yp in addition to Yq, suggesting an alteration in the IR3 region. Among other AZFc candidates, complete TTY3 and partial CDY1 BAC sequences were detected on the proximal 5p and distal 15q regions, respectively, in both the sexes. However, primers deduced from these clones showed male specific amplification of TTY3 and CDY1 exons suggesting (re)organization of their flanking sequences between Y and autosomes. Importantly, approximately 5% males showed CNV of various Y-linked genes, and approximately 3%, random microdeletions across the AZF region. Present study demonstrates hitherto unreported singular structural organization with respect to DAZ, TTY3 and CDY1 genes highlighting organizational complexities of the human Y chromosome in the global context.

6 EFFICIENT TRANSGENESIS IN BOVINE EMBRYOS BY FERTILIZATION WITH ANDROGENETIC TRANSGENIC BLASTOMERES

Pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression is observed in embryos and offspring produced by these techniques. A possible explanation is that the transgene integrates in the embryo genome after the first cell division. Our main objective was to develop a new technique to generate transgenic bovine embryos without mosaic expression and with high efficiency. We hypothesize that fertilizing metaphase II (MII) oocytes with transgenic androgenetic haploidblastomeres (AHB) (from mosaic embryos) would result in non-mosaic transgenic embryos. To this aim, in the first experiment we generated AHB by enucleating IVM MII oocytes, before or after injecting with a single spermatozoon incubated with pCX-EGFP (enhanced green fluorescent protein) plasmid. These treatments were analyzed by Fisher test (P < 0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated before and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116), respectively (P < 0.05). These embryos showed expression of EGFPof 11.8% (4/34) and 42.3% (30/71) (P < 0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analysis. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4 to 16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out by fusing a single AHB to a zona-free MII oocyte, followed by chemical activation. Presumptive zygotes were cultured in SOF medium in the well of the well (WOW) system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them were checked by PCR using Y- and X-specific sequence primers. PCR analysis confirmed Y-specific sequences in all the AHB and XY-specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the expression pattern of Oct-4 (pluripotent marker gene) was examined in the blastocysts by inmunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to that of IVF embryos, indicating efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n = 84). One hundred percent of the embryos showed EGFP expression, with 90.1% non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB is a highly efficient technique for the generation of transgenic non-mosaic bovine embryos.

Buffering of Segmental and Chromosomal Aneuploidies in Drosophila melanogaster

Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF).

Unique Signatures of Natural Background Radiation on Human Y Chromosomes from Kerala, India

BackgroundThe most frequently observed major consequences of ionizing radiation are chromosomal lesions and cancers, although the entire genome may be affected. Owing to its haploid status and absence of recombination, the human Y chromosome is an ideal candidate to be assessed for possible genetic alterations induced by ionizing radiation. We studied the human Y chromosome in 390 males from the South Indian state of Kerala, where the level of natural background radiation (NBR) is ten-fold higher than the worldwide average, and that from 790 unexposed males as control.ResultsWe observed random microdeletions in the Azoospermia factor (AZF) a, b and c regions in >90%, and tandem duplication and copy number polymorphism (CNP) of 11 different Y-linked genes in about 80% of males exposed to NBR. The autosomal homologues of Y-linked CDY genes largely remained unaffected. Multiple polymorphic copies of the Y-linked genes showing single Y-specific signals suggested their tandem duplication. Some exposed males showed unilocus duplication of DAZ genes resulting in six copies. Notably, in the AZFa region, approximately 25% of exposed males showed deletion of the DBY gene, whereas flanking genes USP9Y and UTY remained unaffected. All these alterations were detected in blood samples but not in the germline (sperm) samples.ConclusionsExposure to high levels of NBR correlated with several interstitial polymorphisms of the human Y chromosome. CNPs and enhanced transcription of the SRY gene after duplication are envisaged to compensate for the loss of Y chromosome in some cells. The aforesaid changes, confined to peripheral blood lymphocytes, suggest a possible innate mechanism protecting the germline DNA from the NBR. Genome analysis of a larger population focusing on greater numbers of genes may provide new insights into the mechanisms and risks of the resultant genetic damages. The present work demonstrates unique signatures of NBR on human Y chromosomes from Kerala, India.

Development of Heat-Shocked Tilapia Eggs Following Activation by Water Immersion or Heterologous Fertilization: Implications for the Initiation of Gynogenesis

An attempt was made in this study to initiate artificial gynogenesis in tilapia eggs by water activation, as an alternative to heterologous fertilization. Blue tilapia Oreochromis aureus (Steindachner) eggs activated by water immersion and heat-shocked at 42 oC failed to develop into gynogens. However, heat shock of eggs fertilized with normal or irradiated carp sperm developed into putative gynogens. Un-shocked normal or irradiated carp-fertilized eggs died shortly after hatching due to haploid condition or hybrid inviability. Results of this study indicate that, sperm contact is necessary for successful initiation of gynogenesis. Journal of the Ghana Science Association Vol. 9 (2) 2007: pp. 32-37

AZFc somatic microdeletions and copy number polymorphism of the DAZ genes in human males exposed to natural background radiation

Ionizing radiations are known to induce tumors, chromosomal lesions and minisatellite length variations, yet no correlation has been demonstrated between radiation exposure and indels or copy number polymorphism (CNP) of the genes. We studied the impact of natural background radiation (NBR) on the human Y chromosome owing to its haploid status and clonal inheritance. We analyzed the AZFc region using the DNA from blood and semen of 100 males living near the coastal peninsula in Kerala (India), exposed to NBR along with other 50 normal fertile males. STS mapping of AZFc region showed random microdeletions without conclusive gr/gr or b1/b3 phenotypes. Using a highly specific novel Taqman assay based on sY587 sequence, we detected four copies of the DAZ genes in normal males and 4-16 in those exposed to NBR. Amongst NBR exposed males with multiples copies of the DAZ genes, 75% showed varying FISH signals for DAZ genes with cosmid 18E8 whereas 30% showed mosaicism in terms of presence/absence of the signals in 6-8% cells and unexpected number of signals in 9-12% interphase nuclei. Startlingly, all germline samples studied were found to be free from AZFc microdeletions and CNP of the DAZ genes. Since the DAZ genes are heavily implicated with the germ cell development, the cells with DAZ deletion/duplication are unlikely to survive. Alternatively, an innate mechanism may be operative to protect the germline from the effects of NBR.

Relative efficiency of androgenesis and maize-mediated production frequencies of polyhaploids in winter × spring wheat and triticale × wheat hybrids

Comparisons between androgenesis and maize-mediated haploid production efficiencies were made in six F1 genotypes each of winter × spring wheat and triticale × wheat crosses. The haploid status of the plantlets obtained was confirmed through cytological examination of the root tips. Much higher embryo formation (15.2%), haploid induction (8.7%) and doubled haploid production (8.3%) were obtained in the winter × spring wheat F1s through the wheat × maize system than by androgenesis (3.1%, 3.2 and 2.7%, respectively). Three of the triticale × wheat F1 genotypes failed to respond to androgenesis, while no haploids were recovered through the wheat × maize system in any of the six triticale × wheat F1s. Genotypic specificity, low callus induction and albinism reduced the efficiency of androgenesis both in winter × spring wheat and triticale × wheat hybrids. In all, the wheat × maize system proved to be better for winter × spring wheat hybrids and androgenesis for triticale × wheat hybrids.

Proteome comparative analysis of gynogenetic haploid and diploid embryos of goldfish (Carassius auratus).

Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software. A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa) and 16 protein spots that were up- and downregulated were identified unambiguously, which include some proteins that are correlative with eyes development, nerve development, developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated.

Diploid-dependent regulation of gene expression: a genetic cause of abnormal development in fish haploid embryos.

A diploid-dependent regulatory mechanism of gene expression for spatial patterning of the eye in vertebrates has been determined by analyzing the phenotypes of haploid goldfish embryos. There are two gene loci in charge of eye spatial patterning during embryonic morphogenesis. The expressional probability for each copy of the two genes in a set of chromosomes is 50%. A pair of genes in two sets of homologous or heterologous chromosomes is 100% and essential for normal gene expression. The haploid condition itself would result in the obstruction of gene expression and abnormal development because the diploid-dependent regulatory apparatus will regulate gene expression in a haploid embryo according to the same rule as in the diploid embryo.

O'Neill, S.D., Roberts, J.A. Plant reproduction. Annual plant reviews vol. 6

O'Neill, S.D., Roberts, J.A. Plant reproduction. Annual plant reviews vol. 6

482 In Vivo Micrografts in Almond (Prunus dulcis)

Micrografts have proven a very useful technique when the early propagation of plant material is desired either to invigorate weak material, multiply selected genotypes, or obtained virus-free shoots. This is the situation with the recovery of haploid almond embryos, which occur at low frequency with sexual embryos in twin seeds (i.e., multiple embryos within the same seedcoat). Often these haploid plants show weak growth due to their haploid condition and their poorly developed state within twin seeds. Very little information is presently available, however, concerning the effectiveness of different micrografting techniques for almond. In this work, we examine the success of in vivo micrografting of `Nonpareil' almond seedlings under different conditions. Variables included type of micro-scion, the rootstock genotype, and the growth stage of the rootstock. Microscions tested included small (3 mm) micro-wedges from either unsprouted or recently sprouted buds. Rootstocks evaluated included the `Hanson' (peach × almond) hybrid, and Nemared and Nemaguard peach rootstocks. Rootstocks were grafted after either ≈3 weeks of growth, when the tissue was still herbaceous, or after ≈3 months of growth, when the tissue had become woody. Results show significant differences between the treatments. Findings will be discussed both in terms of effectiveness of different approaches and the advantages and disadvantages of their use in breeding programs.

Variation in Karyotype and Ploidy Level Among Field Isolates of Claviceps purpurea

Karyotype analysis by pulsed‐field gel electrophoresis of several field isolates of Claviceps purpurea showed extensive variation in size and number of chromosomes. Analysis of F1‐ascospore‐lines of a cross between two field isolates showing chromosome‐length polymorphisms (CLPs) indicate that recombination during meiosis contributes considerably to this variability. In addition, cytofluorometrical estimation of DNA content of single nuclei indicate that the C. purpurea strains vary in their ploidy level: most strains probably are ± diploid, some appear to be haploid, others ± triploid (with varying degrees of aneuploidy). Benomyl treatment (as used, e.g. for ‘haploidization’ in the parasexual cycle of imperfect fungi) of some field isolates confirmed that the differences in 4′‐6‐diamidino‐2‐phenylindol (DAPI)fluorescence correspond to different ploidy levels: derivatives with lower nuclear DNA content could be isolated from the putatively nonhaploid strains; a minimal value (even after repeated benomyl treatment) was obtained, corresponding to the lowest value of field isolates, probably representing the haploid status. Most of these strains are not impaired in fitness and aggressiveness and therefore can be used for the selection of recessive mutants and for functional molecular studies.

Prospects for introgressing tomato chromosomes into the potato genome: An assessment through GISH analysis

With a view to assess the possibility of homoeologous pairing and crossing-over between the chromosomes of potato (Solanum tuberosum) and tomato (Lycopersicon esculentum), a somatic fusion hybrid and two monosomic alien tomato addition genotypes were investigated through genomic in situ hybridisation (GISH). The somatic fusion hybrid, C31-17-51, was near hexaploid (2n = 6x - 4 = 68) possessing 46 potato chromosomes + 20 tomato chromosomes + 2 translocated chromosomes. The two alien addition genotypes were near tetraploids (2n = 4x + 1 = 49) and consisted of monosomic alien additions for tomato chromosome 1 in genotype 2103-1, and tomato chromosome 8 in genotype 2301-2. In the fusion hybrid the tomato pachytene chromosome identification revealed that the chromosomes 1, 2, 5, 6, 7, 10, and 12 were in diploid condition whereas among those that were in haploid condition, three could be identified viz., 4, 9, and 11. The remaining three chromosomes could not be cytologically identified. Although the chromosomes with translocated segments could not be identified at the pachytene stage due to technical difficulties, there was clear evidence for the presence of a reciprocal translocation observed at diakinesis and metaphase I stages. Because of autosyndetic pairing of the translocated segments, it gave a false impression as if there was a high frequency (86.0%) of allosyndetic pairing. In contrast to the fusion hybrid, the two alien monosomic addition genotypes showed a very low frequency of allosyndetic pairing, namely 1.1 and 1.3% respectively for the monosomic additions 1 and 8. In the genotype 2301-2, monosomic addition for tomato chromosome 8, crossing-over between the homoeologous chromosomes was estimated to occur in 0.8% of the meiotic cells investigated. Despite this low frequency of homoeologous pairing and crossing-over, there is a possibility for introgressing tomato chromosomal DNA into the potato genome through intergenomic recombination.Key words: Solanum, Lycopersicon, chromosome additions, GISH, introgression.

Prospects for introgressing tomato chromosomes into the potato genome: An assessment through GISH analysis

With a view to assess the possibility of homoeologous pairing and crossing-over between the chromosomes of potato (Solanum tuberosum) and tomato (Lycopersicon esculentum), a somatic fusion hybrid and two monosomic alien tomato addition genotypes were investigated through genomic in situ hybridisation (GISH). The somatic fusion hybrid, C31-17-51, was near hexaploid (2n = 6x - 4 = 68) possessing 46 potato chromosomes + 20 tomato chromosomes + 2 translocated chromosomes. The two alien addition genotypes were near tetraploids (2n = 4x + 1 = 49) and consisted of monosomic alien additions for tomato chromosome 1 in genotype 2103-1, and tomato chromosome 8 in genotype 2301-2. In the fusion hybrid the tomato pachytene chromosome identification revealed that the chromosomes 1, 2, 5, 6, 7, 10, and 12 were in diploid condition whereas among those that were in haploid condition, three could be identified viz., 4, 9, and 11. The remaining three chromosomes could not be cytologically identified. Although the chromosomes with translocated segments could not be identified at the pachytene stage due to technical difficulties, there was clear evidence for the presence of a reciprocal translocation observed at diakinesis and metaphase I stages. Because of autosyndetic pairing of the translocated segments, it gave a false impression as if there was a high frequency (86.0%) of allosyndetic pairing. In contrast to the fusion hybrid, the two alien monosomic addition genotypes showed a very low frequency of allosyndetic pairing, namely 1.1 and 1.3% respectively for the monosomic additions 1 and 8. In the genotype 2301-2, monosomic addition for tomato chromosome 8, crossing-over between the homoeologous chromosomes was estimated to occur in 0.8% of the meiotic cells investigated. Despite this low frequency of homoeologous pairing and crossing-over, there is a possibility for introgressing tomato chromosomal DNA into the potato genome through intergenomic recombination.Key words: Solanum, Lycopersicon, chromosome additions, GISH, introgression.

Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, five new related yeast species from flowers and associated insects.

Five new yeast species, Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, are described to accommodate isolates recovered from flowers and floricolous insects of Australian Hibiscus trees, cosmopolitan morning glories (Ipomoea spp.) and Brazilian cereoid cacti. The new Wickerhamiella species are heterothallic, occur in the haploid condition and are clearly separated reproductively from one another. Although they exhibit little physiological variation, they are easily delineated from Wickerhamiella domercqiae, the only species known previously, by their resistance to cycloheximide and the production of strong extracellular lipases. C. drosophilae and C. lipophila share the latter property, but unlike the Wickerhamiella species, they fail to utilize nitrate as sole nitrogen source. PFGE indicates that these yeasts have an unusually low number of chromosomes. The large-subunit rDNA (D1/D2) sequences demonstrate a close relationship between the five species and Candida vanderwaltii and Candida azyma. Their relationship with W. domercqiae is more distant, but all share, with some other Candida species, a single monophyletic clade. The type and isotype strains are as follows: W. australiensis strains UWO(PS)95-604.3T (h+; CBS 8456T) and UWO(PS)95-631.3I (h-; CBS 8457I); W. cacticola strains UFMG96-267T (h+; CBS 8454T) and UFMG96-381I (h-; CBS 8455I); W. occidentalis strains UWO(PS)91-698.4T (h+; CBS 8452T) and UFMG96-212I (h-; CBS 8453I); C. drosophilae UWO(PS)91-716.3T (CBS 8459T); and C. lipophila UWO(PS)91-681.3T (CBS 8458T).

Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy

Based on the results of animal studies, round spermatid injection (ROSI) has been introduced into the clinical practice of several in-vitro fertilization (IVF) centres. The efficiency of this procedure in terms of fertilization rates and pregnancy rates, however, remains very poor. An essential aspect which does not receive enough attention is the correct identification of this type of round cell within a heterogeneous population of testicular cells. A Nikon inverted microscope equipped with phase-contrast optics (DLL) provided a clear image which allowed reliable recognition of round spermatids in cell suspensions smeared at the glass bottom of the dish. Fluorescent in-situ hybridization confirmed the haploid status of the selected cells. However, exploration of several biopsies from patients with non-obstructive azoospermia showing no spermatozoa after extensive search did not reveal any round spermatids. This observation questions whether enough effort is spent on searching for mature spermatozoa or late spermatids. Experimental investigations should precede the introduction of ROSI into the clinical practice of any IVF centre.

Determination by flow cytometry of the chromosome doubling capacity of colchicine and oryzalin in gynogenetic haploids of gerbera

In vitro plants of the gynogenetic haploid line 86122/560 of gerbera were treated with colchicine or oryzalin dissolved in dimethylsulfoxide, to compare the antimitotic efficiency of these substances. The ploidy level was evaluated by flow cytometry two months after the treatment. Decrement of the multiplication rate was taken into account for the evaluation of the toxic effect of the antimitotic substances. Controls both with and without dimethylsulfoxide maintained the haploid status. At comparable doses, oryzalin proved to be as efficient as colchicine, but slightly less phytotoxic. Longer oryzalin treatments could probably induce the diploidization of a larger number of cells and reduce the problem of chimaeric plants.