Determination Of Ploidy Level Research Articles

Morphological, reproductive and karyological variability in Allium oleraceum in Lithuania

Morphological, reproductive and karyological variability were investigated in fifteen populations of Allium oleraceum transferred from natural populations in Lithuania to the field collection of medicinal and aromatic plants of the Institute of Botany of the Nature Research Centre in Vilnius. Nine populations were tetraploid (2n = 4x = 32), four pentaploid (2n = 5x = 40) and two consisted of both cytotypes. The greatest differences among the populations and between the ploidy levels were observed in stem height and mass of aerial bulbils per plant. The seed production was very low and did not differ significantly between the ploidy levels, while neither tetraploids nor pentaploids did produce any seed if insects were prevented from visiting their flowers. The means of the morphological characters were higher in pentaploids than in tetraploids. However, none of them provided the possibility of determining ploidy level without chromosome counting.

Glycerol-treated nuclear suspensions—an efficient preservation method for flow cytometric analysis of plant samples

Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.

Nuclear DNA content variation associated with muscle fiber hypertrophic growth in fishes

Muscle fiber hypertrophic growth can lead to an increase in the myonuclear domain (MND), leading to greater diffusion distances within the cytoplasmic volume that each nucleus services. We tested the hypothesis that hypertrophic growth in the white muscle of fishes was associated with increases in the mean DNA content of nuclei, which may be a strategy to offset increasing diffusion constraints. DAPI-stained chicken erythrocytes standards and image analysis were used to estimate nuclear DNA content in erythrocytes and muscle fibers from 17 fish species. Mean diploid (2C) values in fish erythrocytes ranged from 0.78 to 7.2pg. Erythrocyte 2C values were used to determine ploidy level in muscle tissue of small and large size classes of each species. Within each species, mean muscle fiber diameter was greater in the large size class than the small size class, and MND was significantly greater in larger fibers for 11 of the 17 species. Nuclear DNA content per species in muscle ranged from 2 to 64C. Fiber-size dependent increases in ploidy were observed in nine species, which is consistent with our hypothesis and indicates that endoreduplication is occurring during fiber growth. However, two species exhibited significantly lower ploidy in the larger size class, and the mechanistic basis and potential advantage of this ploidy shift is unclear. These results suggest that increases in ploidy may be a common mechanism to compensate for increases in MND associated with fiber hypertrophy in fishes, although it is likely that other factors also affect ploidy changes that occur in muscle during animal growth.

Determination of ploidy level by flow cytometry and autopolyploid induction in cocoyam (Xanthosoma sagittifolium)

A procedure for chromosome doubling of the white cocoyam (Xanthosoma sagittifolium ) type was established by using colchicine and oryzalin treatments to in vitro plantlets. Flow cytometry was successfully used for analyzing ploidy levels within three cocoyam types and regenerated plants. Treating in vitro white cocoyam with 0.05% colchicine for 7 days produced tetraploids. The oryzalin treatment (0.05% for 3 days) produced only mixoploids, but it has in turn, a lower rate of mortality. Therefore, in white cocoyam, oryzalin could constitute an alternative to the use of colchicine, whenever the selection and stabilization of polyploids induced by this reagent will be achieved. Key words : colchicine, oryzalin, flo w cytometry, ploidy level, tetraploid, mixoploid, cocoyam (Xanthosomasagittifolium).

Effects of CXCR1 and CXCR2 inhibition on expansion and differentiation of umbilical cord blood CD133+ cells into megakaryocyte progenitor cells

ObjectiveThere have been various reports on the roles of CXC receptors (CXCR) in modulation of hematopoiesis. In the present study, we investigated the effects of CXCR1 and/or CXCR2 inhibition on expansion and differentiation of umbilical cord blood (UCB) CD133+ cells into megakaryocytic progenitors. Materials and methodsPurified UCB CD133+ cells were cultured in a serum-free liquid culture either in the presence or absence of neutralizing anti-CXCR1 and/or anti-CXCR2 antibodies in combination with a conventional cytokine cocktail for up to 14days. Expression of megakaryocytic lineage markers (CD41 and CD61) and determination of ploidy level were determined by flowcytometry. In addition, colony-forming unit assay was performed using CD133+ cultures in serum-free collagen-based medium containing the cytokine cocktail plus neutralizing CXCR1 and -R2 antibodies. Colony forming unit-megakaryocyte (CFU-MKs) and non-MKs were counted after immunocytochemistry staining on day 12. ResultsWe show that while simultaneous inhibition of both CXCR1 and -R2 causes a significant reduction in the fold expansion of UCB CD133+ cells, it also leads to an increase in percentages of CD61+, CD41+, and CFU-MK populations. ConclusionCXCR1 and CXCR2 play significant roles in the suppression of megakaryopoiesis. We demonstrate that blocking of this suppressive effect by a simultaneous inhibition of both receptors can enhance the differentiation of UCB CD133+ cells into megakayocytic progenitors.

Ploidy level determination within the context of in vitro breeding

Variations in genome size and chromosome complement of species provide very useful information for biosystematic studies, and also because they influence a range of ecological characteristics. They are also of utmost importance for breeding, especially when in vitro biotechnology tools are used and the need arises to assess the trueness-to-type of regenerated plants. Thus, protocols have existed for a long time for chromosome counting, and more recently also for the determination of the relative nuclear DNA content and genome size. It has also been shown that these latter traits are strongly correlated to regeneration competence and, more generally, to developmental processes in plants. This article will briefly review such approaches from a methodological and breeding-oriented viewpoint.

Image cytometric measurements of diploid, triploid and tetraploid fish erythrocytes in blood smears reflect the true dimensions of live cells

Comparative image cytometry of erythrocytes of diploid and triploid tench Tinca tinca L. and evolutionary tetraploid sterlet Acipenser ruthenus L. was performed on whole live unstained cells, live cells with stained nuclei and on stained fixed whole cells and their nuclei to test if erythrocyte measurements made from blood smears reflect the true dimensions of live cells. Nuclear area and perimeter were the best ploidy level predictors distinguishing accurately among live and fixed diploid, triploid and tetraploid cells, without significant differences between live and fixed cells within a ploidy level. Redundancy analysis revealed insignificant marginal effect of fixation (explained 2.3% of variation, F = 0.804), whereas the effect of ploidy level was highly significant (explained 50.6% of variation, F = 34.874). The erythrocyte measurements of diploid, triploid and tetraploid fish erythrocytes and their nuclei made from blood smears reflect the true dimensions of live cells, and the fixation procedure did not substantially affect their predictive value for ploidy level determination.

Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x × 2x sexual hybridisation and its application to extensive breeding programs

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x x 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x x 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders' rights.

‘Safor’ Mandarin: A New Citrus Mid-late Triploid Hybrid

World mandarin production rose from 18.3 million tons to 27.9 million tons between the years 2000 and 2007 (FAO, Food and Agriculture Organization, 2009). In Spain, 87% of mandarin production in the last 5 years has been destined to the fresh fruit market (Intercitrus, Interprofessional Citricola Espanola, 2008), which demands high-quality, seedless fruits throughout the marketing season. Therefore, the production of seedless varieties is very important. Triploid plants are generally considered an evolutionary dead end, because they generally give rise to aneuploid gametes with very low fertility (Otto and Whitton, 2000). Predominantly trivalent and a high number of bivalent and univalent associations are formed during meiosis of citrus triploid hybrids (Frost and Soost, 1968). Moreover, abortion of the megasporogenesis in the period between the embryo-sac first divisions and the fecundated egg cell is common (Fatta Del Bosco et al., 1992). For this reason, citrus triploid hybrids are generally sterile, although they can occasionally produce fruits with very few seeds and induce seed formation in fruits of other cultivars. Citrus triploid hybrids can be obtained by means of 2x · 2x hybridizations through the production of unreduced gametes by the female parent (Esen and Soost, 1971). The frequency of unreduced gamete formation depends on the genotype (Esen and Soost, 1971; Luro et al., 2004). Triploid embryos are preferentially found in seeds between onethird and one-sixth smaller than normal seeds and these small seeds generally do not germinate under conventional greenhouse conditions. Embryo rescue from these small seeds is required to reach high germination rates (Navarro et al., 2002). Luro et al. (2004) proposed that Second Division Restitution is the mechanism controlling unreduced gamete formation in clementines, whereas in sweet orange, Chen et al. (2008) proposed First Division Restitution to be the mechanism involved. In these two genetic mechanisms, only one part of the maternal heterozygosity is transmitted to the triploid hybrid and the rate of maternal heterozygosity varies among the loci in relation with the rate of single crossing over between the centromere and a given locus (Ollitrault et al., 2008). Ploidy level determination by histological methods is too laborious for large-scale analyses. However, ploidy level can be accurately determined relatively rapidly in large populations by flow cytometry (Ollitrault and Michaux-Ferriere, 1992). Embryo rescue and flow cytometry are two indispensable techniques for extensive triploid citrus breeding programs. These techniques allow the efficient recovery of plants from embryos contained in small seeds and enable the ploidy level of regenerated plantlets to be determined quickly and easily with just a small piece of leaf while the plants are still in the test tube (Navarro et al., 2002). In Spain, there are numerous problems associated with the production season of mandarin cultivars, which include satsumas [Citrus unshiu (Mak.) Marc.], clementines (C. clementina Hort. ex Tan.), and mandarin hybrids. Satsumas and clementines are traditionally harvested from the beginning of September until mid-February. Satsumas produce seedless fruits because they have sterile pollen and ovules. Clementines, the most widely grown mandarins in Spain, are self-incompatible and also produce seedless fruits if grown in isolation. However, their pollen and ovules are viable, and consequently they are able to pollinate and be pollinated by other sexually compatible cultivars. As a result of the demand for lateseason mandarins by international markets, several midand late-maturing mandarin hybrids were introduced in Spanish citriculture. These hybrids are self-incompatible, but their pollen and ovules are viable and crosspollinate with clementines, producing fruits with seeds in both groups of cultivars, which causes substantial economic losses. With a view to solving this problematic situation, a triploid breeding program was started in Spain in 1996. The main objective was to produce new midand late-maturing triploid cultivars through sexual hybridization, embryo rescue, and ploidy analysis by flow cytometry (Navarro et al., 2002). Recently we released the late-maturing triploid variety Garbi [(C. clementina·C. tangerina) · (C. reticulata · C. sinensis)] (Aleza et al., 2010), which reaches optimum maturation during the second half of March. This variety of mandarin is replacing ‘Fortune’ mandarin (C. clementina · C. tangerina), which reached a peak production of 300,000 tons but is currently being replaced rapidly as a result of its high susceptibility to Alternaria alternata. Another problem is the low fruit quality of ‘Hernandina’ clementine, our latest maturing clementine, when grafted on ‘Carrizo’ citrange (Citrus sinensis · Poncirus trifoliata), which is by far the most predominant rootstock. Fruit peel deteriorates quickly after mid-January, and in practice, no clementine fruits are available by February. In this article, we describe a new triploid hybrid named ‘Safor’ mandarin [(C. clementina · C. tangerina) · (C. unshiu · C. nobilis)] characterized by its high quality, seedless fruits, and mid-late ripening season.

Analysis of breeding systems, ploidy, and the role of hexaploids in three Hypericum perforatum L. populations

Hexaploid seeds are produced by predominantly tetraploid populations of Hypericum perforatum, but the fate of hexaploid seedlings and their reproductive behavior have not been closely examined. We used flow cytometry to analyze single seeds and individual plant samples of three accessions of H. perforatum to determine ploidy levels and reproductive pathways. Seed samples of all three accessions were facultative apomicts, with tetraploid cytotype predominant (85–91%) and a lower frequency of hexaploids (9–14%), with diploids (5%) detected in only one population. Seedling populations consisted of tetraploids (87–97%) and hexaploids (3–13%). Hexaploid embryos are most likely generated by a 2 n gamete of the tetraploid and fertilized by a normal, reduced tetraploid male gamete. These hexaploids are expected to produce unbalanced gametes because they possess chromosome complements that include two triploid sets originally derived from two different species. The observation that some tetraploid seeds had endosperm with high cellular DNA content indicates that some unbalanced male gametes produced by hexaploids were evidently viable and could effect fertilization. Whether this mechanism is also true for egg cells or whether the hexaploids are capable of producing unreduced embryo sacs is uncertain. Because of severe reproductive difficulties, hexaploid seedlings may play a very minor role in gene flow and the further evolution of H. perforatum. The likelihood that hexaploids will evolve to types with an increased frequency of bivalent paring in meiosis is relatively low. However, hexaploids may include novel chemotypes, which could be vegetatively propagated if valuable, medicinal types can be identified among them.

Apomixis in the Sugar Beet Reproduction System

Research on five lines of sugar beet with a tendency towards apomixis showed the presence of facultative apomicts among the studied plants (2.4%). Facultative agamospermy was detected by isozyme analysis and by nuclear DNA amount estimation using flow cytometry. Genetic segregation according to isozymes in seed progenies showed the presence of meiotic agamospermy; its probable mechanism was normal meiosis in tetraploid cells of the female archespore. The occurrence of cytologically unreduced male gametophytes was confirmed in 21% of the plants by the indirect method of determining ploidy level from the number of pore regions in mature pollen grains. These studies identified homozygotic sublines with a tendency towards apomixis, proposed for practical breeding at the Kutno Sugar Beet Breeding Company Ltd., to be included in breeding experiments as components of heterotic hybrids.

PRODUCTION OF GYNOGENIC TETRAHAPLOID PLANTS IN STRAWBERRY THROUGH CULTURE OF UNPOLLINATED OVARIES

The present study was conducted in the Tissue Culture Lab., Department of Vegetable Crops, Faculty of Agriculture, CairoUniversity, Giza, Egypt, during the period from 2005 to 2008. The objective of this work was to produce tetrahaploid plants from strawberry (Fragaria × ananassa Duch.) by culturing unpollinated ovaries of two different genotypes, viz., Camarosa and Sweet Charlie in vitro. Floral buds, 2-4 days before anthesis, were pre-treated at 4˚C for different periods and cultured on different media composition. Germinated ovules were performed and multiplication was induced to produce new shoots. Shoots were rooted and produced whole plantlets for adaptation. Results showed that cold pre-treatment was not efficient for producing the best ovule regeneration and production of tetrahaploid plants. Both cultivars gave nearly similar results under the optimal conditions for ovule regeneration. Determination of ploidy level was carried out and examinations showed that plantlets produced from the ovule culture were tetrahaploid. These results represent a modern tool for breeders to produce tetrahaploid strawberry plants to develop new cultivars in a short period (few months), or using the protocol of ovule regeneration in genetic engineering experiments by transferring new genes to develop better yield or quality of strawberry cultivars.

Crossability, Cytogenetics, and Reproductive Pathways in Rudbeckia Subgenus Rudbeckia

Rudbeckia L. are valuable nursery crops that offer broad adaptability and exceptional ornamental merit. However, there is little information on interspecific and interploid crossability and ploidy levels of specific cultivars. The objectives of this study were to determine the ploidy levels and relative DNA contents (genome sizes) of selected species and cultivars, to evaluate self-compatibility and crossability among species and ploidy levels, and to explore reproductive pathways in triploid R. hirta L. with the goal of facilitating future breeding endeavors and development of new hybrids. Reciprocal interspecific crosses were performed between R. hirta cultivars and R. fulgida Ait., R. missouriensis Engelm. ex C.L. Boynton & Beadle, and R. subtomentosa Pursh. as well as reciprocal interploid crosses among four R. hirta cultivars. A combination of relative DNA content analysis and chromosome counts was used to test for hybridity and to determine ploidy levels for selected species, cultivars, and interploid R. hirta F1 hybrids. Of the specific clones tested, R. subtomentosa and R. missouriensis were diploid, R. fuligida varieties were tetraploid, and R. hirta include both diploid and tetraploid cultivars. Mean 1Cx DNA content varied over 320% among species. The interploid R. hirta crosses produced triploids as well as pentaploids and hexaploids. Seedlings from open-pollinated triploid R. hirta appeared, based on diverse phenotypes and DNA contents, to be aneuploids resulting from sexual fertilization, not apomixis. Of the 844 seedlings from interspecific F1 crosses, only one individual, R. subtomentosa ×R. hirta, had a DNA content intermediate between its parents and was confirmed as the only interspecific hybrid. Although most taxa had low self-fertility, seedlings (with genomic sizes similar to their maternal parent) resulted after interspecific crosspollination, indicating that pseudogamy is one reproductive pathway in Rudbeckia species.

Taxonomy of Mexican and Central American Members of Solanum Series Conicibaccata (sect. Petota)

Members of Solanum series Conicibaccata in Mexico and Central America are very similar. All are tetraploids (2n 5 4x 5 48). Recent authors have recognized three or four species: S. agrimonifolium, S. woodsonii, S. longiconicum (some- times included in the next), and S. oxycarpum. We had difficulty distinguishing these species in the herbarium, and needed to resolve species boundaries for ongoing floristic studies. We studied this group in the field throughout Mexico, Guatemala, Costa Rica, and Panama, grew collections in the greenhouse, studied herbarium specimens, determined ploidy levels through flow cytometry, and generated molecular data using Random Amplified Polymorphic DNA. Molecular data distinguish S. agrimonifolium, S. longiconicum ,a ndS. oxycarpum. Solanum woodsonii was not available for molecular analysis. All four species can be distinguished morphologically, but only by leaf character states that overlap in range, by pubescence differences that are best observed in living specimens, and by a seed spot character that is only evident on living or recently gathered specimens.

Short Communication. A robust method for chromosome quantification and ploidy determination in woody species

Accurate determination of ploidy level of putative polyploid plants is essential for tree breeding and other applications. Methods for ploidy determination include quantification of chromosome numbers in root-tip cells via light microscopy and indirect assessment via anatomical and morphological traits. Flow cytometry is potentially a high-throughput method to quantify nuclear DNA content; however, it does not allow determining chromosome numbers and interfering compounds often prevent its use. Microscopy-based quantification of chromosomes in active root-tip cells remains the most unambiguous method for ploidy determination, although root tips are difficult to obtain from field-grown plants, and light microscopy can result in insufficient resolution in species with many and small chromosomes. Here, we present a robust technique that uses 2, 4-diamidino-2-phenylindole (DAPI) dye and 1000-fold magnification fluorescence microscopy for quantification of chromosomes in root and shoot tips of woody angiosperms and gymnosperms, and overcomes the reported difficulties. Rather than the conventional tip squashing, spreading tips on glass slides resulted in very good chromosome separation in diverse species, with up to 56 chromosomes and a chromosome size of 2–20 μm. Chromosome counts were performed in diploid Agathis robusta, Elaeocarpus angustifolius, Eucalyptus robusta, Paulownia tomentosa, Pongamia pinnata and Toona ciliata, and di- and tetraploid Acacia crassicarpa and Citrus species.

Is triploidy indisputably determinable in hybridogenetic hybrids by planimetric analyses of erythrocytes?

The size of amphibian erythrocytes is positively correlated to their DNA content. Therefore, planimetry of erythrocytes has long been used to determine ploidy levels in amphibians. In Rana esculenta. a hybrid originating from Rana ridibunda x Rana lessonae natural crosses, planimetric determination of erythrocyte size is used to distinguish diploid and triploid specimen. In the present study we performed planimetric analyses on the hybrid Rana grafi, comprising a second hybridogenetic system with its parental species Rana ridibunda and Rana perezi in southern parts of France. We found size differences of diploid and triploid erythrocytes to range between 26 and 33% in studies on adult R. esculenta and calculated a mean difference of 30%. Our results strongly support a significant influence of the factors snout-vent-length (SVL), geographic location, organic matter of substratum, and relative amount of oxygen on the erythrocyte size of these hybrids. Discriminant analyses proved a probability of approximately 30% to disaffiliate individuals in our data set and indicated that ploidy determinations on individuals will be most unprobable, if the SVL of individuals strongly staggers and the habitats differ in their environmental characteristics. Due to the found dependencies of the erythrocyte size to several factors we consider planimetric analysis for reasons of ploidy determination as unprobable and thus we favour more sophisticated techniques such as DNA-flowcytometry or karyotype analyses.

Polyploidy in the Olive Complex (Olea europaea): Evidence from Flow Cytometry and Nuclear Microsatellite Analyses

Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives. Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance. Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana. This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors of subspp. guanchica and europaea.

Controlled Pollination with Sorted Reduced and Unreduced Pollen Grains Reveals Unreduced Embryo Sac Formation in Diospyros kaki Thunb. ‘Fujiwaragosho’

This study investigated ploidy levels of endosperm and embryos in seeds of the hexaploid (2n = 6x = 90) Diospyros kaki ‘Fujiwaragosho’ to determine the possible origin of unusual ploidy levels observed in seedlings of this cultivar. Female flowers of ‘Fujiwaragosho’ were pollinated with sorted reduced (3x) and unreduced (6x) pollen grains of ‘Zenjimaru’ in 2004 and 2005. Embryo rescue culture was conducted with normally developing seeds that were excised from immature fruit harvested in August. Ploidy levels of the endosperm and plantlets from the embryos in the seeds were determined by flow cytometry. In some cases, chromosomes of root tip cells of the plantlets were also counted to determine ploidy levels of the embryos. It appeared that seeds obtained with reduced pollen pollination contained a normal hexaploid embryo and nonaploid (9x) endosperm, while those obtained with unreduced pollen pollination mostly contained a dodecaploid (12x) embryo and octadecaploid (18x) endosperm. These results indicated unreduced embryo sac formation in ‘Fujiwaragosho’. We discuss the possible cause of the occurrence of only nonaploid and octadecaploid endosperm, respectively, with reduced and unreduced pollen pollinations.

CUCUMBER (CUCUMIS SATIVUS L.) DIHAPLOID LINE PRODUCTION USING IN VITRO RESCUE OF IN VIVO INDUCED PARTHENOGENIC EMBRYOS

Homozygous dihaploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions, source of resistance to diseases, would be useful to accelerate breeding for new resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollination with pollen irradiated at 0.5 KGy with a Co 60 γ-ray source; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized under a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μM colchicine. Rescue of diploid or chimerical plants was confirmed by flow cytometry, previous to their acclimation and plant growth in the greenhouse. Selfing of colchicine-treated haploid plants allowed the perpetuation by seed of homozygous lines. Seeds were harvested from almost all lines after selfing in the greenhouse. The high rate of seed production facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation, and accelerate breeding for new commercial varieties. DHLs have been used as parental lines for the production of commercial hybrids.

Estimation of Relative Nuclear DNA Content in Dehydrated Plant Tissues by Flow Cytometry

The power of flow cytometry in field plant research may be greatly enhanced by analysis of nonfresh tissues. Previous attempts to use chemical fixatives, however, received only little attention because of protocol complexity and limited time, after which successful assays were achieved. This unit describes simple and rapid procedures for relative nuclear DNA content estimation in dehydrated tissues of vascular plants by DAPI flow cytometry. Histograms with reasonable resolution can be obtained in several-year-old specimens. The approach here is particularly suitable for reliable determination of DNA ploidy level, although detection of small variation in nuclear DNA content is also possible in many cases. Retrospective ploidy inference in silica-dry or herbarium vouchers and simplified transport of plant material from remote areas are among the principal benefits for plant biosystematics, ecology, and population biology.