Cytophotometric Analysis of the DNA of the Nucleus

Cytophotometric results of DNA analyses were already introduced when talking about the doubling of the amount of DNA during S-phase. The following measurements have to be taken into account when dealing with comparative quantitative measurements of the amount of nuclear DNA:

	The DNA of nuclei has to be stained specifically. The Feulgen-reaction proved to be especially useful for this purpose. It is based on the binding of colourless leucofucin (an alkaline fuchsin made colourless by treatment with sulphurous acid) to aldehyde residues rendering a red colour complex. The required aldehyde groups are the result of a previous hydrolytic treatment of the DNA. Standardized experimental conditions (controlled temperature, concentration of the reactants, length of hydrolysis, duration of the reagent’s influence) are strictly to be followed.
	Staining does despite the utmost experimental care never produce absolute, reproducible experiments. The error rate is very high. The results of each measurement have therefore to be related to an intern control, a so-called standard. Usually, diploid nuclei of the root tip of Allium cepa containing 33.5 pg DNA [1pg = 1 picogram = 10-12 g] are used.
	The choice of the nuclei (cells) has to be made with great care, since polyploidization is very common during ontogenesis and tissue specialization and the amount of DNA doubles during each S-phase of the cell cycle. It is therefore common to analyze nuclei of the telophase of meristematic tissue.
	The resulting amount of DNA refers to the haploid or the diploid genome (n or 2n). The values are called (1)C- or 2C-values.

Measurements of stained nuclei occur in the cytophotometer, a device consisting of a microscope, a photometer, and an integrator. Cytofluorometry during which the nuclei are fluorochromed is even more sensitive than cytophotometry. DAPI (4’,6-diamino-2-phenylindol) or Hoechst 33258 (2-[2-(4-hydroxyphenyl-) 6-benzimidazolyl- 6-(l-methyl, 4-piperazyl) benzimidazol]) are especially well-suited as specific fluorochromes.

If the experimental conditions are chosen and standardized correctly, the amount of fluorescence generated due to complex formation between DNA and fluorochrome is directly proportional to the amount of DNA. Only few results have been gained by this method yet.

The 2C-values of a number of gymnosperms and angiosperms are known. The angiosperm numbers cover more than three magnitudes, while that of gymnosperms vary by a factor of 15 only. The extremes can vary between 1:10 and 1:50, polyploidy being just one of the causes.

Relatively few measurements of algae, mosses, and ferns have been conducted, but it is already becoming quite clear that the average values of these groups are below that of the evolutionary higher developed angiosperms. When comparing the species with the smallest amounts of DNA of each class, a clear increase in the amount of DNA with increasing evolutionary development is apparent.

Amount of DNA per cell or per organelle. In some viruses: amount of RNA. An increase of the DNA with rising evolutionary development becomes apparent (according to A. H. SPARROW, H.J.PRICE, A.G.UNDERBRINK, 1972).

The introduced values show that considerable differences in the DNA-content of their genomes exist within many genera despite the large similarities of their species. This phenomenon could at first not be explained, it was therefore termed paradox of the C-value. We know today that the larger part of the DNA of all eucaryotes is non-encoding and this part can change in size from species to species without effecting active genes. We will treat this phenomenon in more detail in the next section.

The amounts of DNA correlate primarily with the degree of ploidy, while differences in the size of the genome are often secondary causes for evolutionary changes. The values of species with several ploidy races behave therefore often not as integer multiples of a basic amount. More often, the increase of the amount of DNA as a function of the degree of ploidy results from a logarithmic function, a saturation curve.

Some striking correlations connected to the evolution of plants are nevertheless obvious. Specializations of angiosperm genera are either correlated with a decrease of the amount of DNA like in many Scilla-species or both cases occur: in some species the amount decreases wile it increases in others (Microseris-species).

Microseridinae: Changes in the amount of DNA in the course of adaptive radiation. 1. and 2. perennial species: Microseris, 3. Phalacroseris, 4. an annual Microseris, 5. a perennial Agroseris, 6. an annual Agroseris (H. J. PRICE and K. BACHMANN, 1975).

The amounts of DNA are usually higher in gymnosperms than in woody angiosperms. The highest values were detected in Pinus-species and cycads. M.D.BENNETT established the following rules after an analysis of 271 herbaceous angiosperms:

1. Annual species have on average significantly less DNA than perennial species. This is true for both monocots and dicots.

2. Diploid annual species display only very little variation of the amount of DNA, while perennial species – many of which are polyploid - show large variations.

3. Ephemeral annual species i.e. species going through the whole life cycle starting with germination and ending with dissemination in just a few weeks with flowers that last only one day, have on average usually less DNA than annuals with non-ephemeral flowers.

4. The DNA-content of obligatorily perennial monocots is significantly higher than that of facultative perennials. No significant difference between facultatively perennial and annual species exist.

These correlation lead to the assumption that the amount of plant DNA (the nucleotype) correlates with their duration of development. Annual species do usually develop very quickly, the cycles of mitosis follow each other rapidly, meiosis is short. These conditions can only be met with little DNA per nucleus.

Species with a lot of DNA are thus ‘necessarily’ perennial in regions with a short duration of vegetation. A perennial life style does nevertheless not necessarily cause large amounts of DNA: some perennials contain only very small amounts of DNA.

In 1985, J.P. GRIME and his collaborators (University of Sheffield) pointed out that the amount of DNA and the amount of information contained by the genome are independent values, and that the amount of DNA correlates with the beginning of shoot and leaf growth. All spring-flowering plants on North-English meadows are characterized by a large amount of DNA and an extensive leaf growth primarily based on the elongation of already existing tissue primordia. As a consequence, these species acquire an ecological advantage in still cold seasons. Species with small amounts of DNA have another timing: their growth, a consequence of cell division, occurs at a later, more favourable season, elongation does not play the dominant role it has with the spring-flowering plants.