Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species.

Received: 20 March 2007 Returned for revision: 14 May 2007 Accepted: 8 June 2007 Published electronically: 7 August 2007†Background and Aims After the initial boom in the application of ﬂow cytometry in plant sciences in the late 1980sand early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts weremade to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers areutilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plantDNA ﬂow cytometry.†Methods GPB and WPB were used to prepare samples for ﬂow cytometric analysis of nuclear DNA content in a setof 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemicalcomposition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidiumiodide ﬂuorescence, coefﬁcient of variation of DNA peaks, quantity of debris background, and the number of par-ticles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using thebuffer that performed better for a given species.†Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis ofsamples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution thanthose prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performedbetter and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histogramsin comparison with previously published buffers was achieved in most of the species analysed.†Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species.Although GPB failed with some plant species, it provided high-quality DNA histograms in species from whichnuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPBor WPB is suitable for preparation of high-quality suspensions of intact nuclei suitable for DNA ﬂow cytometry.Key words: Cytosolic compounds, ﬂow cytometry, general purpose buffer, genome size, lysis buffers, nuclear DNAcontent, nuclear DNA staining, propidium iodide, woody plant buffer.

TECHNICAL ARTICLE Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

Coriander, Coriandrum sativum L.

Karyotype analysis provides insights into genome organization at the chromosome level and into chromosome evolution Chromosomes were marked for comparative karyotype analysis using FISH localization of rDNA genes for the first time in Apioideae species including taxa of economic importance and several wild Daucus relatives Interestingly, Daucus species did not vary in number of rDNA loci despite variation in chromosome number (2n = 18, 20, 22, and 44) and previous publications suggesting multiple loci All had single loci for both 5S and 18S-25S (nucleolar organizing region) rDNA, located on two different chromosome pairs The 5S rDNA was on the short arm of a metacentric chromosome pair in D crinitus (2n = 22) and D glochidiatus (2n = 44) and on the long arm of a metacentric pair in other Daucus species, suggesting possible rearrangement of this chromosome For other Apiaceae, from two (Apium graveolens), to three (Orlaya grandiflora), to four (Cuminum cyminum) chromosomes had 18S-25S rDNA sites Variability for number and position of the 5S rDNA was also observed FISH signals enabled us to identify 20-40% of the chromosome complement among species examined Comparative karyotype analysis provides insights into the fundamental aspects of chromosome evolution in Daucus

https://bsapubs.onlinelibrary.wiley.com/doi/pdf/10.3732/ajb.0700007

Major cytogenetic landmarks and karyotype analysis inDaucus carota and other Apiaceae

Extensive karyotype analysis including determination of somatic chromosome number, total chromosome length and volume and estimation of 4C DNA amount were carried out on 9 different cultivars of ginger (Zingiber officinale Rose.) for the first time. A significant variation in nuclear DNA amount was recorded at the cultivar level. The 4C DNA amount varied from 19.663-24.102 pg in the cultivars studied. The correlation coefficient studies showed that the 4C DNA content and genomic chromosome volume were interdependent. The structural alteration of chromosomes as well as loss or addition of highly repetitive sequences in the genome showed variation in the DNA amount at cultivar level, but a marginal variation in nuclear DNA content at the cultivar level indicated a close relationship between them.

/pdf/estimation-of-4c-dna-and-karyotype-analysis-in-ginger-1yed20fmes.pdf

Estimation of 4C DNA and Karyotype Analysis in Ginger (Zingiber officinale Rosc.) -I

Extensive karyological analysis including determination of somatic chromosome number, total chromosome length and volume and estimation of 4C DNA amount were carried out on 8 different varieties of Coriandrum sativum L. A significant variation in nuclear DNA amount was recorded in the varietal level. The structural alteration of chromosome as well as loss or addition of highly repetitive sequences in the genome have played a vital role in intervarietal DNA variation. The absence of wide differences in nuclear DNA content in the varietal level indicate a close relationship between them.

Anathbandhu Das

Papers

Variation in 4C DNA Content and Chromosome Characteristics in Different Varieties of Coriandrum sativum L