Estimation Of Nuclear DNA Content Research Articles

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

AbstractFusion experiments were performed with a first (BC1‐6738) and a second (BC2‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC2 genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC1‐6738 or BC2 genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC1‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC2‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.

Nucleus–Cytosol Interactions—A Source of Stoichiometric Error in Flow Cytometric Estimation of Nuclear DNA Content in Plants

Discrepancies of 2–20% were noted in nuclear DNA content estimates using mixed and unmixed extracts of several plant species combinations (target/standard). This may be because cytosolic components affect dye accessibility to DNA. Several dilution experiments showed clearly how cytosol concentration modified dye accessibility. Heat treatment suggested that dye accessibility could be related to chromatin sensitivity to decondensation. Some methods (nucleus isolation, dilution) can decrease, but not eliminate, stoichiometric error. The high sensitivity of the nucleus to the medium highlights the problem of reliability limits in estimating genome size. The choice between internal and external standardization and the interpretation of intraspecific variation in ‘nuclear DNA content’ are discussed.

Evaluation of the nuclear DNA content and GC percent in four varieties of Fagus sylvatica L

The nuclear DNA content of Fagus sylvatica has been assessed for the first time by flow cytometry and propidium iodide staining. Three beech varieties were compared to the common beech: the tortuosa, the purpurea and the pendula varieties. Values were 2C = 1.11 ± 0.02, 1.11 ± 0.01, 1.12 ± 0.01 and 1.13 ± 0.01 pg, respectively. These are the first estimates of nuclear DNA content made in the Fagus genus. GC percent was estimated in the common beech and in the tortuosa variety with mithramycin.

Comparative Karyomorphology and DNA Estimation Studies in Ginger Cultivars (Zingiber officinale Rosc.).

Chromosome studies and in situ estimation of nuclear DNA content were carriedout in two different cultivar of Z. officinale Rosc. Somatic chromosome number 2n=22 was found to be constant in both the cultivars. Karyomorphological analysis indicated high heterogenity. DNA content of these two cultivars were highly varied and a correlation could be obtained between the amount of 4C DNA and the total chromosome length. Average packing ratio was workedout from which the relation between the 4C nuclear DNA content and the total chromosome length were established.

Intertribal somatic hybridization between rapid cycling Brassica oleracea L. and Camelina sativa (L.) Crantz

Black spot, caused by Alternaria brassicae and A. brassicicola, is an important disease in all Brassica oleracea vegetables. Sufficient resistance to the pathogen is not found within the species, nor in species that readily cross to B. oleracea. Camelina sativa (false flax) is highly resistant to Alternaria spp. and has, in addition, other desirable characters for the improvement of B. oleracea. Protoplast fusions were performed between rapid cycling B. oleracea (tribe Brassiceae), which has good regenerability, and C. sativa (tribe Sisymbrieae) by polyethylene glycol (PEG) treatment. The B. oleracea fusion partner was inactivated by treatment with iodoacetate. C. sativa has poor regenerability; hence, no pretreatment was needed for this species. The protoplasts were cultured using a feeder layer system. A total of 2903 calli were isolated from the fusions. Fourteen of these initiated shoots, i.e., 0.5% regeneration frequency. Approximately 110 shoots were excised from 6 of these calli and transferred to rooting medium. Rooted plantlets grew vigorously in vitro and flowering was frequently observed. However, establishment of rooted shoots in soil was unsuccessful. Hybrid identity was confirmed by intermediate shoot morphology, RAPD marker analysis, and flow cytometric estimation of nuclear DNA content.

Comparative flow cytometric estimation of nuclear DNA content in oil palm (Elaeis guineensis Jacq) tissue cultures and seed-derived plants.

Flow cytometric analysis performed on two different crosses of dura×pisifera oil palm gave an accurate estimation of nuclear DNA content. The genome size of Elaeis guineensis was found to be 2C=3.76±0.09 pg and therefore ca. 3.4×109 bp. Embryogenic calli and plants showed the same ploidy level, but the measured 2C DNA values differed significantly. No variation in the ploidy level between three different types of calli originating from foliar explants, namely nodular compact callus, fast-growing callus and friable callus was observed. Since fast-growing callus (FGC), already identified as a source of `mantled' phenotype variants, did not show any difference in their ploidy level, these results are consistent with the hypothesis of an epigenetic origin for this type of somaclonal variant.

Reliable flow cytometric estimation of nuclear DNA content in coffee trees

Flow cytometry gave high resolution of genome size in two coffee taxa (Coffea liberica dewevrei and C. pseudozanguebariae). Propidium iodide (PI) and Petunia hybrida were used as dye and internal standard, respectively. Proportionality between the DNA content and the digitized fluorescence signal was checked. Five main results were evident. First, optimal experimental conditions were established for peak location estimation (mean or mode), staining time (at least 2 minutes), high voltage (557 V) for the photomultiplier tube (PMT), and PI concentration (333 μg/ml). Second, a parameterization of the effects of high voltage and PI concentration were deduced from curve fitting. Third, two biases in DNA content estimation were recorded for high voltage and PI concentration, and were minimized. Fourth, the genome sizes of C. liberica dewevrei and C. pseudozanguebariae were estimated with accuracy 2C = 1.421 ± 0.005 pg and 2C = 1.129 ± 0.005 pg, respectively. Fifth, between-genotype variance was emphasized within each taxon. © 1996 Wiley-Liss, Inc.

Reliable flow cytometric estimation of nuclear DNA content in coffee trees

Flow cytometry gave high resolution of genome size in two coffee taxa (Coffea liberica dewevrei and C. pseudozanguebariae). Propidium iodide (PI) and Petunia hybrida were used as dye and internal standard, respectively. Proportionally between the DNA content and the digitized fluorescence signal was checked. Five main results were evident. First, optimal experimental conditions were established for peak location estimation (mean or mode), staining time (at least 2 minutes), high voltage (557 V) for the photomultiplier tube (PMT), and PI concentration (333 micrograms/ml). Second, a parameterization of the effects of high voltage and PI concentration were deduced from curve fitting. Third, two biases in DNA content estimation were recorded for high voltage and PI concentration, and were minimized. Fourth, the genome sizes of C. liberica dewevrei and C. pseudozanguebariae were estimated with accuracy 2C = 1.421 +/- 0.005 pg and 2C = 1.129 +/- 0.005 pg, respectively. Fifth, between-genotype variance was emphasized within each taxon.

Transfer of resistance to Xanthomonas campestris pv campestris into Brassica oleracea L. by protoplast fusion

Black rot caused by the bacterium Xanthomonas campestris pv campestris is one of the most serious diseases of Brassica oleracea. Since sources of resistance to the disease within B. oleracea are insufficient and control means are limited, the development of resistant breeding lines is extremely desirable. Certain lines of B. napus contain very high resistance controlled by a dominant gene, but crossing the two species sexually is very difficult. Therefore, somatic hybrids were produced by protoplast fusion between rapid cycling B. oleracea and a B. napus line highly resistant to X. campestris pv campestris. Hybrid identity was confirmed by morphological studies, flow cytometric estimation of nuclear DNA content, and analysis of random amplified polymorphic DNA (RAPD). Inoculations with the pathogen identified four somatic hybrids with high resistance. The resistant hybrid plants were fertile and set seed when selfed or crossed reciprocally to the bridge line '15' (Quazi 1988). Direct crosses to B. oleracea were unsuccessful, but embryo rescue facilitated the production of a first-backcross generation. The BC1 plants were resistant to the pathogen. Progeny from the crosses to 'line 15' were all susceptible. Embryo rescue techniques were not obligatory for the development of a second-backcross generation, and several resistant BC2 plants were obtained.

Novel flowering and fatty acid characters in rapid cycling Brassica napus L. resynthesized by protoplast fusion

Novel rapid cycling Brassica napus lines have been produced by protoplast fusion between rapid cycling B. oleracea and rapid cycling B. rapa. Fusion products were selected based on iodoacetate inactivation and regeneration ability. A total of 36 plants was recovered from 3 regenerating calli. All were confirmed as somatic hybrids by morphological features, flow cytometric estimation of nuclear DNA content, RAPD analysis and/or DNA hybridization. Plants from two of the calli contained chloroplasts from B. rapa, and plants from the third contained B. oleracea chloroplasts. Some plants flowered in vitro, but on average flowering was initiated 22 days after transfer to soil. Although seed set was fairly low after self pollination, more seeds were obtained from pollination of open flowers than from pollination of buds. Seeds of the somatic hybrid B. napus showed novel fatty acid compositions, different from the mean of the two parental lines. Flowering was monitored in plants grown from seeds of the somatic hybrids, rapid cycling B. napus (CrGC 5-1) and the two diploid parental genotypes. Progeny of the somatic hybrids flowered faster and were more vigorous than rapid cycling B. napus (CrGC 5-1). The improved lines contain chloroplasts from B. rapa, unlike rapid cycling B. napus (CrGC 5-1), which has B. oleracea chloroplasts. The somatic hybrid lines produced may be useful for genetic studies or further in vitro manipulations.

Nuclear DNA content of commercially important Eucalyptus species and hybrids

This paper reports the nuclear DNA content estimates obtained by flow cytometry for a group of twelve Eucalyptus species and five fast-growing hybrids that includes those most widely planted throughout the world. Estimates of nuclear (2C) DNA content for the species surveyed ranged from 0.77 pg/2C for Eucalyptuscitriodora Hook. (subgenus Corymbia) to 1.47 pg/2C for Eucalyptussaligna Smith (subgenus Symphyomyrtus). This range corresponds to a haploid genome size range of 370–700 megabase pairs. The average physical equivalent of a 1 cM distance could be as low as 200 kilobase pairs in Eucalyptus, an attractive feature for positional cloning efforts in woody plants. The closer the species were in phylogenetic relationship the more similar were their nuclear DNA content values. All the interspecific hybrids surveyed displayed a nuclear DNA content in the expected intermediate range between the respective parental species, with the exception of one originating from Rio Claro, Brazil, whose exact parentage is unknown. No evidence of polyploidy was observed in any of the hybrids. The flow cytometry procedure employed in this study is an efficient method for investigating ploidy levels of high yielding hybrids of Eucalyptus.

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4′,6‐diamidino‐2‐phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species‐specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Karyological and cytophotometric study of expiant derived clones of non-polysomatic and polysomatic species ofKniphofia

Cytological and cytophotometric analysis of root tips of regenerated plants, derived from rhizome expiant ofKniphofia nelsonii andKniphofia uvaria, revealed marked difference in behaviour of chromosomes and level of 4C nuclear DNA content. Karyotypic stability could be retained in all 52 regenerants of K. nelsonii whereas inK. uvaria out of 75 regenerants analysed, 12 plants were exclusively diploid and the rest 63 plants were predominantly diploid comprising variable amount of aneuploid and tetraploid cells. Cytological data was further confirmed by nuclear DNA content estimation. Alteration in the structure of chromosomes could also be noted in 57 regenerants ofK. uvaria giving rise to two new karyotypes. The use of polysomatic tissue for securing variantsin vitro inK. uvaria has been suggested.

Estimation of nuclear DNA content of plants by flow cytometry

Estimation of nuclear DNA content of plants by flow cytometry

Estimation of nuclear DNA content of plants by flow cytometry

A rapid and simple protocol for estimation of nuclear DNA content of plants is described. Suspensions of intact nuclei are prepared either by chopping plant tissues or lysing protoplasts in a MgSO4 buffer, mixed with DNA standards, and stained with propidium iodide in a solution containing DNAase-free RNAase. Fluorescence intensities of the stained nuclei are measured by a flow cytometer. Values for nuclear DNA content are estimated by comparing fluorescence intensities of the nuclei of the test population with those of appropriate internal DNA standards. The same procedure can also be used for rapid determination of ploidy in plant tissues.

Estimation of nuclear DNA content by flow cytometry in fishes of the genus Xiphophorus

1. 1. By use of flow cytometry we measured nuclear DNA content in cells from 16 stocks representing 9 species of the genus Xiphophorus. 2. 2. Significant differences were detected between certain stocks and species with respect to DNA content. 3. 3. Male-female differences were apparent in 5 of 7 stocks in which males and females were studied. 4. 4. Estimation of nuclear DNA content is of potential significance in connection with the genetics of sex determination and the study of taxonomic relationships.

Simultaneous Feulgen densitometry and autoradiographic grain counting with the Quantimet 720D image-analysis system. I. Estimation of nuclear DNA content in 3H-thymidine-labeled cells.

Free accessAbstractFirst published January 1982Simultaneous Feulgen densitometry and autoradiographic grain counting with the Quantimet 720D image-analysis system. I. Estimation of nuclear DNA content in 3H-thymidine-labeled cells.R J SklarewVolume 30, Issue 1https://doi.org/10.1177/30.1.7054272