Bull. Amer. Iris Soc. 158: 25-33 (1960)
Iris Colors and Pigments
Dr. Peter Werckmeister

Can one still expect new colors in Iris? This question concerns not only the breeder, but also the amateur who takes pleasure in viewing the colors of the many new varieties appearing every year. The popularity of the flamingo pinks which spread so rapidly among irisarians is clear proof of how much more important a new color is than other progress in breeding.

The origin of this new color is known. As the result of a mutation a new pigment, not found in any wild iris, made its appearance. This new pigment, lycopin, in turn produced other color combinations with already existing types of' pigments. However, all of them have in common the tangerine beard.

If, therefore, at some future time in additional pigment hitherto unknown in the genus Iris should be discovered there would be the possibility for additional new iris colors. May we expect additional new pigments in iris? Is there a possibility that wholly new iris colors may make their appearance?

The attempt to anticipate such future events is like a voyage of discovery in an unknown country, and such a voyage requires careful preparation if one is to keep his feet on solid ground. It is not a question of designing tapestry flowers without real life. Therefore, it is necessary to be well informed about all possible conditions underlying the wide diversity of flower colors. The next step is to find out which of these occur in iris. Then it is essential to determine how colors not found in iris have made their appearance in other garden flowers. Only in this manner can one arrive at a conclusion concerning the possible origin of new colors in iris.

In the past, few colors appeared unexpectedly and then it took all of the breeders' skill to combine this new attribute with other qualities to create superior garden varieties. The sweet pea is an example of this. Formerly there was no scarlet sweet pea, but suddenly a new pigment appeared by mutation and sweet peas of bright scarlet color were produced. Today we have a comprehensive knowledge of flower pigments, permitting prophecies concerning what may be expected and suggestions as to how these expectations may be realized in iris. Let us have a look at what is known at present.

The Distribution of Color in Iris Flowers

When a section of the petal of an iris flower is examined under the microscope it is surprising to find that nearly always the color pigment is found only in the thin epidermal layer, and the underlying parenchyma cells show no color in ordinary light. It is only in the ultraviolet range that fluorescent pigments may be seen there; but these must not be neglected since, as copigments, they alter the appearance of the visible pigments. It is very astonishing that nature produces the most saturated colors in an extremely thin epidermal layer of pigment.

All flowers with a velvet sheen when viewed under the microscope are seen to have definitely protruding, papillate epidermal cells. Where there is no velvet sheen these papillate cells are not observed. Velvety dark violet pansies have such epidermal cells, but the dull black pansies have rounded, non-protruding cells. Although the color of these pansies is different they have the same kind and quantity of pigment; the difference in color is due to the difference in the form of their epidermal cells.

A velvet sheen and highly concentrated pigment can be seen in deep violet irises, in a most beautiful manner in the signal spot of Iris susiana, and that is where the papillae are most elevated. Where the epidermal cells of the flower of I. susiana are flat the color is a dull gray. The velet sheen is most important with irises of dark color. For example, the falls of variegatas and amoenas are deeper in color if the velvety sheen is present, whereas the yellow and white colors of the standards do not lose their brilliancy if the sheen is lacking.

Cell Sap and Plastid Pigments

When the pigment-carrying epidermal cells of the iris flower are viewed through the microscope it can be seen that there are two kinds of pigments localized in different parts of the cell. One kind, the anthocyanins which are chymochrome pigments, produces colored cell sap (Figure 1a). The other kind are the yellow carotenoid pigments, found only in plastids, which are tiny biscuit-shaped structures in the cytoplasm of the cell (Figure 1b). In gray, brown or red-purple flowers both occur in the same cell (Figure 1c).

In the petals and sepals (standards and falls) of tangerine pinks the lycopin pigment is found in the plastids; and the same is true of the tangerine-colored beard of these flowers. Thus the beautiful shell pinks are white-diluted tangerine. This being so we can understand why the tangerine pigment can be combined in the same flower with all the known shades of antliocyanin; each is in a different part of the pigment-carrying epidermal cell. However, it is sometimes difficult to recognize the pink color if it occurs with yellow plastid color.

Now I have found that it is easy to extract the yellow pigment with alcohol, but lycopin is insoluble in alcohol and is not removed. If then orange-colored beards are immersed in alcohol the yellow color is removed and they turn pink if they contain lycopin; if no lycopin is present they become white. Also, plants heterozygous for the tangerine gene, which appear among F2 progenies and are of Tit, genetic constitution, can be detected by this test. Thus such tests are important for the breeder or geneticist because in some irises with bright orange beards the color is due entirely to orange colored carotenoids.

Reds to Blues Due to Anthocyanins

The cell sap pigments mainly are the bearers of colors between red and blue. It is the anthocyanins and their copigments that produce color in this range. Many anthocyanins are known, all derived from basic anthocyanidins, and their chemical structure is of interest because a slight change in the structure of the molecule may cause a change in color. At the present time 8 anthocyanidins have been found in garden flowers (Figure 2). All are derived from the same basic molecular structure and are known chemically as sugar-free aglycones. When a change in flower color appears, either as the result of mutation or segregation, the new color usually represents a change to a chemically simpler molecule.

If we examine the colors of garden flowers it is found that certain anthocyanidins are present in scarlet flowers, others occur in gentian blue flowers, whereas most of them overlap in the intermediate purple and violet range.

Anthocyanins and their copigments nowadays can be identified by means of paper chromatography. With this technique chemical substances in solution are concentrated on absorbent paper at localized points and may be identified by their appearance and relative positions. This display of chemical substances on paper is called a paper chromatogram (Figure 3). Two-dimensional chromatograms as we prepare them show very nicely the differences in the pigments of diverse iris colors. New anthocyanins can be recognized easily and we are able to identify various colors with definite pigments and combinations of pigments. There are six principal causes of color variation between red and blue, which will now be considered.

Fig. 1. Diagrammatic representation of papillate epidermal cells from iris flowers of different color: a, from a violet flower with colored cell sap and colorless plastids, indicated by cross-hatching of central part of cell and few surrounding plastid granules in black; b, from a yellow iris with colorless cell sap and yellow plastids, indicated by absence of cross-hatching and additional plastid granules; c, from the purple falls of a variegata flower with colored cell sap and yellow plastids.

Causes of Red to Blue Color Variations

1. Variation in pH of cell sap. This can be shown readily in vitro. If acid is added to a test tube containing anthocyanin extract the mixture turns red; if alkali is added it turns blue, and then often very rapidly green. This result must be ascribed to the presence of copigments which change from colorless to yellow when alkali is added. Willstaetter found this same cyanin in red roses and blue cornflower and believed that the change in color was due to the pH effect. Now we know that the pigment of the blue cornflower is an aluminum-iron-chelate of cyanin. The acidity of the cell sap in the living cell can be assumed to vary within narrow limits (precise pH measurements are problematical in the living cell). Consequently color changes in nature due to differences in pH are not so great as formerly believed.

2. Variation in the chemical structure of anthocyanidins. There are known today eight anthocyanidins. The most complicated of these occur in wild flowers, whereas in garden flowers they may change by mutation into simpler forms. The chemical formulae of these anthocyanidins are shown in Figure 2, with the arrows indicating the direction of the change from the more complex to the simpler molecules.
    Most scarlet flowers contain pelargonidin, the simplest anthocyanidin. I know of only one exception, the scarlet cyclamen, which contains paeonidin; but scarlet peonies equally owe their color to pelargonidin.
    All gentian blue flowers have delphinidin except the blue cornflower, Centaurea, which has cyanidin. Delphinidin is the principal iris anthocyanidin and the iris anthocyanins derived from it differ mainly in sugar and organic acid content.
    Hirsutidin, the most complicated anthocyanidin has been found up to now only in Primula hirsuta. Rosinidin has been found in Primula rosea. It is a triumph of the chromatogram technique that rosinidin was found and classified, although it has not yet been isolated and identified by classical methods.
    The colors of the anthocyanidins on the acid chromatograms show what is to be expected in the red series. They form three color groups according to their molecular structure: delphinidin, petunidin, malvidin and hirsutidin are purple; cyanidin, paeonidin and rosinidin are carmine; pelargonidin is scarlet.
    The fuchsia flower is the most instructive example of color variation in different parts of the flower corresponding to the anthocyanins present—paeonidin is found in the calyx and malvidin in the corolla.
    Of the greatest interest are the garden flowers, such as the cinerarias, which contain all of the pigments in the anthocyanin range. These teach us that blue colors are due to delphinidin only, violets to delphinidin and cyanidin, carmines to cyanidin only and, finally, the scarlets to pelargonidin only. Comparison of the chemical formulae shows that this progression from blue to red involves a change to chemically simpler molecules.
    It can be predicted that a really red iris will not be found until we discover a cyanidin or pelargonidin mutant. A comparison of our so-called red irises with the truly red flowers of other garden plants shows that in iris the combination of delphinidin and yellow carotenoids only produces a grayish red or brown. In pansies cyanidin is found and the combination gives a more brilliant color.
    Since the direction of color mutation preferably leads to a chemically simpler molecule it is not wholly improbable that one day a truly red iris with cyanidin or pelargonidin will be found. This could happen among garden seedlings, especially after inbreeding. But it is also possible that crossing to a wild species with any other anthocyanidin might lead to a greater variation in the formula of the anthocyanidins.
    The greatest advance towards red that can be obtained with delphinidin is realized in Linum grandiflorum rubrum. If additional progress is to be made it will be necessary to look for a new anthocyanidin.
    Many years ago, Hayashi isolated a malvidin glycoside from an iris and called it ensatin after Iris ensata, which is the name applied to I. kaempferi in Japan. My own search for new kinds of anthocyanidins led me to detect malvidin, first in I. chrysographes and later in an Abbeville red seedling of a Louisiana iris which was received from Mr. Steiger. Therefore, breeders of apogons may have the best chance of obtaining a color break. However, I have found malvidin in the signal spot of the oncocyclus species I. auranitica and in a rather red form of a sofarana-like iris found with the assistance of Professor West near Laklouk in Lebanon.

Fig. 2. Chemical formulae of anthocyanidins found in garden flowers, arranged in order of complexity, as they appear on 2-dimensional paper chromatograms, with the arrows indicating the direction of change from the more complex to the simpler molecules. The position of OH groups able to form aluminum chelates are marked with heavier lines in the formulae of petunidin, delphinidin and cyanidin.

3. Variation of the glycosides. In Primula sinensis Scott-Montcrieff found that strains with diglucosides had bluer colors than those with monoglucosides. I have found other glycosides in Cayeux's diploid tall bearded iris variety FLORIDOR which transmitted its new anthocyanin to its seedlings. Whether the new glycosides in iris will produce new colors cannot be predicted at present. The dwarfs also have interesting possibilities. The sugars found thus far in iris anthocyanins are glucose, xylose and rhamnose.

4. The copigment effect. This, in our present state of knowledge, is of the greatest importance for understanding the variations from red to blue in garden irises. In examining the chromatogram of a red-purple iris like DISPLAY (Figure 3) it is significant that it shows fewer copigment spots than does the chromatogram of a blue iris like BLUE RHYTHM (Figure 3). This effect can be shown very nicely in a test-tube experiment. If the pigments of iris flowers are extracted with very dilute hydrochloric acid the extract of BLUE RHYTHM, even in this acid solution, is perceptibly bluer than that of DISPLAY. At such a degree of acidity, the pH effect no longer can be effective. If to the extract of DISPLAY is added the extract of a white iris like GUDRUN, the color changes immediately to the same hue shown by the extract of BLUE RHYTHM. From this it is apparent that the copigment effect, not the pH in this highly acid solution, is responsible for the color change.
    My chromatograms show that there are copigments differing in kind and quantity that are characteristic for different color groups. Some are derived from the colored isoflavon irigenin, but there are others perceptible mostly by a strong fluorescence in ultraviolet light. With orchid pinks most anthocyanins and copigments are much reduced, but here one notes the cumaric acids which are held to be a first step to anthocyanins.

5. The chelate effect. If we aim at progress in the blue range of iris colors, the chelate effect is of special interest. Chelates are definite chemical compounds in which a metallic ion combines with an organic molecule at two positions having the appearance of a pair of tongs or a chela. These positions are marked with heavier lines in the formulae of delphinidin, cyanidin and petunidin in Figure 2. Trivalent aluminum, for example, forms chelates only with organic molecules with three hydroxyl groups of which two must be adjacent to each other. Therefore, of all the known anthocyanidins only delphinidin, petunidin and cyanidin are able to form aluminum chelates. The chelates are stable in weak acid solutions such as the cell sap of living flower tissues, and remain in the colloidal state.
     With the exception of Centaurea and perhaps other Compositae the true blue colors of other known blue flowers are due only to delphinidin. Bayer showed that there exists more than a single stable aluminum and iron chelate of cyanin with different bases. This explains why cyanin chelates in nature can be true blue as in Centaurea cyanus.
    Aluminum as an ion is soluble only in acid solution. Therefore, roots take it up only from acid soils, not from alkaline soils. That is why hydrangeas grown on alkaline soil are never true blue; they must be cultivated on an acid soil to have true blue color.
     It has never been proved that blue iris colors are due to chelates but it is highly probable. The color of iris anthocyanin chelates prepared in vitro at pH 3 are much bluer than the blue iris flowers known in nature. Since cultivated violas are bluer than wild ones, so with iris further progress towards genuine spectral blue can be expected.
     Of all the varieties I know the old ULTRA, in its standards, goes farthest into the blue, the standards being very nearly true gentian blue. Noteworthy also in this respect are the flowers of the Reticulata species, Iris histrio var. aintabensis, and it may be significant that Mrs. Stevens reports having grown seedlings of I. munzii on an acid soil in New Zealand which were bluer than the species grown in its natural habitat in California.

6. The dilution effect. This effect is of interest although not causing wide color variation. I discovered this effect by chance when diluting an anthocyanin extract, and noting that in doing so it became not only brighter but equally bluer. On closer examination I found that this is not caused simply by a pH effect or some chemical change, since it can be observed when a bottle with a narrow neck is filled with the extract. The solution appears lighter and bluer in the neck of the bottle.
     The dilution effect is not observed with pure solutions of anthocyanin; it is seen only in a collodial extract. Since cell sap is a colloidal solution this effect is almost certain to play some role in the cell, although it is unlikely that this can be definitely proved.

Now it is very remarkable that, apart from tangerine-colored irises with small amounts of anthocyanin, there is not one iris with lighter anthocyanin-colored standards proving the contrary. All bitones either show the same color tone in standards and falls, or the standards are not only lighter but also somewhat bluer. This effect may explain the fact that the near approaches toward genuine blue have been reached in the lighter blues, for instance SOUTH PACIFIC. In looking for new anthocyanidins it should be borne in mind that, when somewhat diluted, cyanidin will look rather bluer than one might expect.

Fig. 3. The distribution of anthocyanins, orange fluorescing copigments and cumaric acids on 2-dimensional paper chromatograms prepared from the flowers of iris showing varying amounts of these components: upper left, the variety DISPLAY; upper right, the variety BLUE RHYTHM; lower left, I. chrysographes; lower right, the variety FLORIDOR. The anthocyanins are shown in black, the orange copigments are the stippled areas, and the areas outlined in black are occupied by the cumaric acids, The circles at the intersections of the straight lines marking the left and lower limits of each chromatogram are located where the preparation of each chromatogram starts.

The Anthocyanin of White Irises

Finally, something about the white irises should he mentioned, since some contain anthocyanin as a normally uncolored pseudobase, which becomes visible if the flowers are immersed in a very dilute solution of hydrochloric acid. In former publications in English I have stated that this pseudobase extract then shows a pink fluorescence; but this statement is somewhat misleading, for the effect can be observed in plain daylight and does not have the fluorescent character in the original meaning of the word. On the contrary, with a mineral acid the colorless pseudobase is changed to the visible anthocyanin.

With tetraploid tall bearded varieties the effect is primarily observed with the so-called dominant whites, not with all-whites like MATTERHORN. Equally, diploid recessive whites fail to show the effect. In tests of their pure white flower parts, amoenas show the effect, but not plicatas. Since yellow flowers have no visible anthocyanin it was of interest to test them. Here the test is somewhat hampered by the fact that carotenoid epoxydes turn blue with mineral acids. In general, the intense yellows and the standards of yellow variegatas show the effect, but not the lemon yellows. It must be stressed, however, that this in no way proves that the absence of visible anthocyanin in the standards of amoenas and variegatas is due to a dominant gene. Such a conclusion can only be made if sufficiently large progenies of suitably planned crosses are carefully counted.

It may be of interest that the effect is found in species hybrids having a recessive white as one parent. With other species it is a question of doubt whether the test can be used to determine if a white is dominant or recessive. But it can make clear whether there are white iris species containing an anthocyanin as a pseudobase and others quite devoid of it.

The chemical test is especially positive with varieties having occasionally irregular anthocyanin splashes. Personally, I am of the opinion that it is questionable whether these irregular splashes in all cases can be considered to be due to virus infections as is reported occasionally nowadays. Seedlings from certain crosses always show these spots and splashes, even if the plants are produced by embryo culture and proved healthy in every respect. Crosses with white chamaeirises and arils of all sorts tend to show them. Primarily, this seems to prove that the balance between free anthocyanin and pseudobase is chemically very unstable and can be altered in one direction or the other by the minutest change of the cell's chemical-physical composition. It is surprising that species hybrids involving dominant white tetraploids, at least in former days (then including yellows), tended to show irregular patches.

This leads to another working hypothesis which I would like to propose here for discussion. To my knowledge it has not been definitely proved by precise analysis of Mendelian ratios—which by the way is very difficult in view of the large number of seedlings required—that the dominant white is due to a gene suppressing anthocyanin. On the contrary, this conclusion is drawn solely from the fact that all crosses between two dominant white irises always produced some blues as well as whites, and it has been assumed that the parents must all be heterozygous. It is not, however, indispensable to ascribe this solely to an inhibitor gene localized in a chromosome. The irregular splashes of the early tetraploid tall bearded and of species hybrids seem to indicate another possibility.

There might indeed be a factor localized in the cytoplasm or in the plastids controlling the change from anthocyanin to pseudobase or vice versa. Such a plasmon would not, even in the case of a mitosis, be distributed equally to the daughter cells, so that mosaics might arise from the irregular distribution of the plasmon. There is therefore the possibility of interpreting the irregular splashes as well as the appearance of some blue seedlings as a plasmon effect. It is not possible to delineate briefly here all possibilities to prove one or the other interpretation.

Recessive whites merit our interest as much as the subjects already discussed. We imagine today that the effect of a gene consists in controlling a synthesis by means of a definite enzyme. This should then be valid also for an anthocyanin, which is a complex molecule synthesized only by different steps. Therefore there are several points where synthesis could be blocked through a gene effect failing to take place. From this it may be concluded that there are several different recessive whites, and such indeed exist. They are readily identified if, when crossed perhaps with MATTERHORN, they produce 100 per cent blue seedlings. Now these must be of course chemically distinguishable and one could learn from them a great deal about the synthesis of anthocyanin.

It would be a very good thing if breeders would have different recessive whites for chemical analysis even though some, obtained as seedlings from crosses between two anthocyanin-colored varieties, have no garden value. For enlarging our knowledge of iris pigments such plants would certainly be valuable.

The whites show further that copigments apparently have no decisive influence on the development of yellow colors. This was to be expected since with even slight pH changes to the alkaline side and with the chelate effect they turn yellow.

Summary

If we review our present knowledge of iris pigments it can be summarized as follows.

1. Carotenoids determine the color range from lemon to tangerine.
2. In the red to blue range we find anthocyanins derived from delphinidin which promise, in consequence, color hues from purple to violet to blue.
3. From several species of apogons we have identified malvidin glycosides, but as yet we cannot predict its influence on color.
4. Colors in the blue range of the spectrum are today essentially determined by the copigment effect, although the chelate effect seems to promise us considerably bluer shades.
5. Prospects of obtaining new anthocyanidins by mutation and species crossing must not be underestimated in comparison to other flowers, especially Viola which shows relationships similar to those of Iris.
6. New color mutants have been obtained in other garden flowers mostly through inbreeding. They appear as chance mutations generally leading to a chemically simpler molecule. In iris this suggests an approach to the red color range.
7. Another way to obtain new colors is by species crossing, and we know a number of garden plants in which new pigments and new colors have been produced in this manner. Perhaps crosses with apogon species and oncocyclus hybrids might accelerate progress.

Dr. Peter Werckmeister is professor of botany at the Botanical Institute, Geisenheim, Germany. He maintains interesting collections of irises in the experimental garden of the Institute, and at his residence where oncocyclus species and regelio-cyclus irises were blooming when Mrs. Randolph and I visited the Werckmeisters May 1959. This article was submitted at my request, first in the original German with a translation which I have edited and have had approved by Dr. Werckmeister.—L.F.R.