Detection of Intraspecific DNA Content Variation in Zea mays L. by Flow Cytometry

Flow cytometry was used to determine quantitative intraspecificDNA content variation in Zea mays. Previous studies using flowcytometry had indicated that intraspecific variation in cornwas beyond the resolution of the method. The DNA content variationamong corn lines observed in this study was in agreement withthe amount of variation observed using microdensitometry. Inorder to observe intraspecific variation, the fluorochrome DAPIwas shown to be superior to mithramycin. The fluorochrome: nucleiratio was found to be critical when DAPI was used because ofself absorption of the fluorescence. Flow cytometry with thefluorochrome DAPI was found to be a rapid and reliable alternativeto microdensitometry in examining intraspecific DNA contentvariation in corn. Key words: Genome size, corn (maize), Zea mays, DAPI     

Flow cytometric analysis for ploidy level differentiation of 45 hairy vetch accessions

Determining the ploidy of plant germplasm is a necessary step in breeding or genetic studies in species. The purpose of this research was to determine the presence of ploidy level differentiation of hairy vetch (Vicia villosd) germplasm. Flow cytometry and root tip chromosome squashing methods were employed to assess 45 accessions labeled V. villosa available through the USDA germplasm collection. Flow cytometry determined that 43 of the accessions were 2C, one accession was 4C, and one accession was 6C. Analysis of accessions by root tip chromosome counts indicated that all accessions were diploid. The 2C accession contains 14 chromosomes and their chromosomes were approximately one-half and one-third in size as compared to the chromosomes of the 4C and 6C accessions, respectively. The 4C accession was observed to have 16 chromosomes and the 6C accession was observed to have 14 chromosomes. The large-scale differences in DNA amounts were due to chromosomal size variability as opposed to ploidy differences. This revealed the incidence of species misidentification of these two V. villosa accessions to be Vicia pannonica. All the V. villosa accessions were observed to be diploid and have similar DNA amounts. Flow cytometry proved to be useful in the efficient assessment of these accessions.     

Nuclear DNA Content Diversity in Chinese Soybean Introductions

Intraspecific nuclear DNA content has been documented in variousangiosperm species. The purpose of this study was to determinethe nuclear DNA content variation in soybean (Glycine max(L.)Merr.). Several studies have suggested that DNA content variationexists in soybean. Ninety soybean lines representing diversegeographic locations in China were analysed by flow cytometry.Nuclei were isolated and stained with either the fluorochromeDAPI or PI. After analysis, it was determined that PI stainednuclei more accurately assessed the total DNA content in soybean.A 12% variation in nuclear DNA content was observed among the90 lines. The amount of nuclear DNA in the lines was withinthe DNA range of United States cultivars previously examined.Nuclear DNA content variation in soybean is much less than thevariation reported in maize. These results could be due to thelack of polymorphism in large chromosomal elements containinglarge blocks of repetitive DNA. In addition, unlike maize, theamounts of DNA variation did not decrease as a result of moreintensive breeding in United States cultivars. Intraspecificnuclear DNA content variability is very different between thesetwo major agronomic species. Glycine max(L.) Merr.; germplasm; genome size     

A Lack of Nuclear DNA Content Variability Among Wheat Near Isolines Differing in Aluminium Response

Nucleotypic variation has been speculated to play a role inthe adaptation of crop species to environmental stress. Theobjective of this study was to determine if nuclear DNA contentvariability was associated with aluminium (Al) tolerance inwheat. Six wheat (Triticum aestivumL.) near isolines (differingin Al response), two recurrent parents (Al sensitive), and onedonor parent (Al tolerant) were all analysed for nuclear DNAcontent using flow cytometry. A 1.7% variation in nuclear DNAcontent was observed among the nine wheat lines. No associationbetween Al response and nuclear DNA content was observed. Allof the wheat near isolines had a nuclear DNA content similarto their recurrent parent. The wheat genome appears to be stablewith no unusual inheritance of nuclear DNA content observed.Flow cytometric analysis proved to be sensitive enough to detectnuclear DNA content variability at the level of 0.5% variationamong wheat lines.Copyright 1999 Annals of Botany Company Genome size,Triticum,breeding, near isogenic.     

Intraplant Nuclear DNA Content Variation in Diploid Nuclei of Maize (Zea mays L.)

Diploid nuclei from stem, mesocotyl, nodal root and root tiptissue of two maize hybrids were examined with respect to theirDNA content. The nuclei were isolated and stained with DAPIand passed through a flow cytometer-cell sorter. The titrationcurve for each tissue was determined. Significant variationwas observed among nuclei of different tissue types. Stem androot tips had the highest diploid nuclear DNA amounts while2-week-old mesocotyl had the lowest diploid nuclear DNA amount.These results provide evidence that during plant developmentand differentiation, the amount of DNA within a diploid nucleuschanges through loss of specific DNA sequences. This study alsodemonstrates the sensitivity of flow cytometry in detectingsmall intraplant variation in nuclear DNA. Key words: Flow cytometry, fluorochrome DAPI, DNA content, tissue differentiation, plant development     

Chromosome doubling of the bioenergy crop,Miscanthus×giganteus

The perennial grass, Miscanthus×giganteus is a sterile triploid, which due to its growth rate and biomass accumulation has significant economic potential as a new bioenergy crop. The sterility associated with the triploid genome of this accession requires labor‐intensive vegetative, instead of seed propagation for potential commercial production. Chromosome doubling was used to produce hexaploid plants in an effort to restore fertility to M×giganteus. Tissue culture derived calli from immature inflorescences were treated with the antimitotic agents, colchicine and oryzalin in liquid and solid media. Calli survival rate decreased with increasing concentrations and durations of colchicine or oryzalin treatments and ranged from 0% to 100%. Nuclear DNA content, as determined by flow cytometry, indicated that the frequency of chromosome‐doubled calli varied between compounds and concentrations with the greatest proportion of callus doubling observed using 2‐day treatments of 15 μm oryzalin (78%) or 939 μm colchicine (67%). Liquid media treatments were more effective than solid gels for chromosome doubling. Although oryzalin was effective at chromosome doubling, it inhibited callus growth and plant regeneration frequency. Seven hexaploid plants with doubled DNA content were generated, which displayed increased stomata size (30.0±0.2 μm) compared with regenerated triploid M. ×giganteus plants (24.3±1.0 μm). Following clonal replication these plants will be evaluated for growth rate, biomass accumulation, and pollen viability. Successful chromosome doubling and plant regeneration of M.×giganteus suggests that ploidy manipulation of this plant and its parental species (Miscanthus sinensis and Miscanthus sacchariflorus) could be a means to access genetic variability for the improvement of Miscanthus as a biofuel/bioenergy crop.